Review of the Cyanobacterial Genus Phormidesmis (Leptolyngbyaceae) with the Description of Apatinema gen. nov.

Round 1

Reviewer 1 Report

The manuscript is a review of the phylogenetic classification of Phormidesmis genus of cyanobacteria and proposed a new species, Apatinema gen. nov. based on phenotypic and genotypic analyses.

require editing by a scientific/technical editor proficient in English

word choice, sentence construction

check in text citation format

check significant digit reporting

related sentences can be placed in the same paragraph (example line 51-79)

know the difference between “isolate” and “strain”

please, check Phormidesmis vs Phormisesmis (very few); very confusing; may use P for Phormidesmis and Phs for Phormisesmis

lines 18, 19-20  remove dashes:  Phormidesmis – P. nigrescens, P. pristley P. communis – and

            “… Phormidesmis (P. nigrescens, P. pristley and P. communis) and…”

            “   of a new taxon – Apatinema gen. nov.”:         “…of a new taxon, Apatinema gen. nov.”

line 26  revise:  “Such (like what?) nutritional strategies…”; replace “Such” to make sense of the sentence

            suggestion: Various/Acquired/Multiple/

line 31-32         “In spite of the progress in the cyanobacterial phylogeny, their taxonomy still has unresolved problems.”

            suggestion:       still undergoing revision/modification

line 33  “… thrive in the most type of terrestrial habitats

line 34  “capacity”        you mean capability

line 40  spell-out “…and don’t do not have a resolution”; no contraction in scientific manuscript

line 41  “In r Recent studies based on comprehensive approach, …”

line 52  the word “cryptic” overused;      “…appear to be cryptic.”

            alternate words: ambiguous, equivocal, questionable, vague

lines 55-64        “In the recent study of Phormidesmis [16] two species – P. arctica Raabová, L. Kováčik,

Elster et Strunecký (reference strain CCALA 1101) and P. communis Raabová, L. Kováčik,

Elster et Strunecký (reference strain AB 11-10) – were described and Leptolyngbya nigrescens Komárek was classified as Phormidesmis nigrescens (Komárek) Raabová, L. Kováčik, Elster et Strunecký.”

lines 60-64       “Unfortunately, the description of Phormidesmis arctica and P. communis by [16] are

invalid due to absence of holotypes citation and unpreserved cultures in a metabolically

inactive state that is in accordance with the requirements of the International Code for

Algae, Fungi and Plants [23]. This fact adds confusion to the understanding of Phormidesmis taxonomy.

Suggestion:

Raabová et al [16] described two species of Phosmidesmis (P. arctica, CCALA 1101 and P. communis, AB 11-10) and Leptolyngbya nigrescens classified as P. nigrescens. Their description of P. arctica and P. communis are invalid due to the absence of holotypes citation and a description of unpreserved cultures in a metabolically active state in accordance with the requirements of the International Code for Algae, Fungi and Plants [23].

Lines 66-69 “… in course of phylogenetic traits placed…”

you mean “ based on phylogenetic traits?

            “…thus there are no…” need a comma after “thus”      thus, there are no

not clear what you mean by this, revise:       “… thus there are no common acceptance of the genus and species limits and level of infraspecific variability. stayed unresolved species delimitation within a genus.”

note:     there is a period after variability

line 70-73

            word choice for “reveal”            alternate words: elucidate,

            remove dash and replace with comma in “…proved a description of the and proposed a new genus, Apatinema gen. nov.”

lines 75-76        2. Materials and Methods                                

                        Sampling

                        need more specific info here

                                    How were the samples collected?  Single sample? replicate samples?

                                    sample containers, storage after sampling, storage in the lab?

                                    sampling dates

                        Sampling Sites

            Table 1. Description of sampling sites. localities

              I suggest putting Norway to be consistent since you mentioned Russia country

            you can replace “Number” as # to save space

            can remove periods; replace with comma, instead; use  simple phrases

line 109 -115     “Svalbard’s cyanobacterial flora has been characterized in previously studies [ref]. Samples (Figure 1) were collected at the different territory of the polar deserts of the North East Island have been conducted cyanobacterial diversity: (east coast of Rijpfjorden bay, (#1) [26], Innvika cove coast (#3) [27], Pyramiden (#4) [28] and the west part of Oscar II Land (#5) [29] of West Spitsbergen Island were investigated. The data on the cyanobacterial diversity of the Polar Ural Mountains (Figure 1, plot  #s 8, 9) are given in a recent were reported in previous study [30]. The main characteristics of sampling areas are were listed in cited articles.”

            “…characterized in previously studies (need references).”      

can replace “plot” with #

line 118 “… the samples were cultivated in liquid Z8 medium and BG11 agar plates [31, 32].” Cultures were maintained under artificial light illumination (35 μmol photons m^–2 s^–1), 16:8h light/dark at 22^oC.

line 125      Describe what is “L” information system

lines 126-133    how many cells were analyzed per sample?

                        were the samples a mixture of cyanobacteria? If so, what per cent?

lines 133-138, 146, 206 check in text reference citation

“Amplification of partial 16S rRNA gene and 16S-23S ITS rRNA region was were performed with pair of primers 1 (5’-CTCTGT GTG CCT AGG TAT CC-3’) suggested by [34] and 27F (5’-AGA GTT TGA TCC TGGCTC AG- 3′) suggested by [34, 35]. The internal primer 2 (5’-GGG GGA TTT TCC GCA ATG GG-3’) by [36] was additionally used for sequencing [36].

Move lines 146-147 (last sentence) to line 138 as above.

            line 206 “The trees topologies reconstructed from both datasets were congruent and resembled the phylogeny of Synechococcales as published previously [4].         

line 142             spell-out TAE and EthBr in “… TAE gels by EthBr staining…”

                        ethidium bromide

lines 152-154    revise the convoluted sentence; clarify

lines 223-225   “In course of Based on 16S estimation, our Phormidesmis strains were found in four clades intermingled with accessions from [16] and other GB (what is GB data?) data {16, more references], whereas on topologies from the whole region of 16S rRNA gene and 16S-23S ITS only three clades could be recognized.

line 262            “… more variable in 16S gene loci then than the two…”

lines 263-264       Clarify what you mean “It is robustly diverged from both Phormidesmis nigrescens and P. priestleyi and differed from them in a similar level.”    what do you mean by “differed in a similar level”?

line 279             Taxonomic description

            this section is better on as a table (characteristics/description, habitat, etc.)

line 391            delete “Basically…”

lines 392-393                “…evidence that the species is widespread. characterized by a wide areal.

line 393             check spelling basionym

lines 405-407    remove dashes

line 408             word choice for “evidence”; alternate words showed, revealed

            as in “The ecological properties Phormidesmis nigrescens evidence showed that it is a typical subaerophytic.        

lines 414-431    better as a table

lines 434-435    “Obviously, the morphological data (Table 4) are not reliably unable to distinguish species in Phormidesmis and related ive genera.”

Author Response

Dear reviewer

We appreciate your time and productive suggestions which definitely improved Phormidesmis manuscript. We agree with most corrections, and we included them into the new version of the article. Our changes represented in the table below:

Reviewer 2 Report

In this paper, the authors describe the morphological and genotypic diversity of 23 strains of cyanobacteria isolated from different places in the Arctic zone. They show that these strains belong to three species of the genus Phormidesmis (P. nigrescens, P. pristley, P. communis) and to the new genus Apatinema, which they describe in this manuscript.

In general, the paper makes a positive impression. And the authors have every reason to correctly identify their Phormidesmis  strains and describe the new genus. However, the presentation of the material requires significant adjustments.

1. The use of a large number of 16S and 16S-23S ITS sequences that do not belong to the genus Phormidesmis sensu lato seems redundant (Figs. 2, 3). It does not carry significant information, but significantly enlarges the trees, which makes them more difficult to read (small print, etc.). The emphasis in bold for strains isolated by the authors that are not Phormidesmis-strains (listed in Appendix A) is also confusing. I have no idea why they are needed in the trees and in the manuscript.

The purpose of these trees is to show the apartness of individual genera, as well as species within the genus Phormidesmis. So, I think it would be right to optimize phylogenetic trees: remove all unnecessary strains and use only representatives of Phormidesmis sensu lato (including those incorrectly identified in NCBI if needed) + reference strains of validly described species of neighboring genera (please mark the reference strains on the trees). If the authors consider another principle of strain selection for the trees to be correct, then this should be carefully substantiated.

2. Analisys of 16S-23S ITS regions are used, as a rule, to justify the division of species within a genus. In this case, a comparison of the secondary structures of D1-D1', V2 and boxB -helixes are usually taken as a basis. Phylogenetic trees constructed with 16S-23S ITS sequences are used, as a rule, as an auxiliary analysis. Why did the authors prefered tree construction rather than secondary structure analysis? Perhaps it is worth drawing secondary structures for the strains isolated by the authors + for the reference strains of three Phormidesmis species. 16С-23С ITIS is used, as a rule, to justify the division of species within a genus. In this case, a comparison of the secondary structures of individual hairpins is usually taken as a basis. Trees built according to ITS sequences are used, as a rule, as an auxiliary analysis. Why did the authors prefer tree construction rather than secondary structure analysis? Perhaps it is worth drawing them for the strains isolated by the authors + for the reference strains of three Phormidesmis species? Or please justify the absence of such a need.

3. For some reason, the morphological description of the isolated Phormidesmis strains appears in the Discussion section, and not in the Results (Figs. 5, 6, 8, Table 3 and and related text). And it would be correct to add the morphological data for reference strains of each Phormidesmis species to Table 3 (for comparison with the isolated strains).

4. Fig. 7. It would be good to analyze here the geographical distribution of representatives of all three Phormidesmis species, and not just P. nigrescens. I wonder if the Arctic and Antarctic strains are really the same species? To discuss this issue, it would be very appropriate to analyze the pupative secondary structures of the 16S-23S ITS regions. Although, additional analyzes may be needed to answer this question (for example, house-keeping genes rpoB, gyrB etc., or ideally ANI comparison). But this is the maximum task. Although it is certainly extremely interesting, because we are talking about the concept of a species in cyanobacteria and the distribution areas of separate species (are there cosmoplite species?). Of course, within the framework of this manuscript, the authors may not solve this problem, but it would be interesting to mention and somehow discuss it.

Extensive editing of English language and style required!

Fig. 8 - There is no figure capture

Author Response

Dear reviewer,

We appreciate your time and productive suggestions which definitely improved manuscript. We agree with most corrections, and we included them into the new version of the article. Our changes represented in the text below

1. The use of a large number of 16S and 16S-23S ITS sequences that do not belong to the genus Phormidesmis sensu lato seems redundant (Figs. 2, 3). It does not carry significant information, but significantly enlarges the trees, which makes them more difficult to read (small print, etc.). The emphasis in bold for strains isolated by the authors that are not Phormidesmis-strains (listed in Appendix A) is also confusing. I have no idea why they are needed in the trees and in the manuscript.

The purpose of these trees is to show the apartness of individual genera, as well as species within the genus Phormidesmis. So, I think it would be right to optimize phylogenetic trees: remove all unnecessary strains and use only representatives of Phormidesmis sensu lato (including those incorrectly identified in NCBI if needed) + reference strains of validly described species of neighboring genera (please mark the reference strains on the trees). If the authors consider another principle of strain selection for the trees to be correct, then this should be carefully substantiated.

We made a lot of different preliminary variant of trees. The Phormidesmis sensu lato tree is not correct, because we get the short branches and couldn't reconstruct the phylogeny.

2. Analisys of 16S-23S ITS regions are used, as a rule, to justify the division of species within a genus. In this case, a comparison of the secondary structures of D1-D1', V2 and boxB -helixes are usually taken as a basis. Phylogenetic trees constructed with 16S-23S ITS sequences are used, as a rule, as an auxiliary analysis. Why did the authors prefered tree construction rather than secondary structure analysis? Perhaps it is worth drawing secondary structures for the strains isolated by the authors + for the reference strains of three Phormidesmis species. 16С-23С ITIS is used, as a rule, to justify the division of species within a genus. In this case, a comparison of the secondary structures of individual hairpins is usually taken as a basis. Trees built according to ITS sequences are used, as a rule, as an auxiliary analysis. Why did the authors prefer tree construction rather than secondary structure analysis? Perhaps it is worth drawing them for the strains isolated by the authors + for the reference strains of three Phormidesmis species? Or please justify the absence of such a need.

We added secondary structures in a manuscript, but we desagree with the reviewer that the those one could be a more significant that primary nucleotide sequences, and the phylogeny. Different methods of phylogeny trees use statistical techniques for inferring probability distributions to assign probabilities to particular possible phylogenetic trees. Those approaches recovers a tree that represents the most likely clades. Secondary structures could given an additional  phylogenetic signal.

3. For some reason, the morphological description of the isolated Phormidesmis strains appears in the Discussion section, and not in the Results (Figs. 5, 6, 8, Table 3 and and related text). And it would be correct to add the morphological data for reference strains of each Phormidesmis species to Table 3 (for comparison with the isolated strains).

We are consider the morphology and distribution of taxa and don't present our result only. It is a compilation of recent rewisions and our notes.

4. Fig. 7. It would be good to analyze here the geographical distribution of representatives of all three Phormidesmis species, and not just P. nigrescens. I wonder if the Arctic and Antarctic strains are really the same species? To discuss this issue, it would be very appropriate to analyze the pupative secondary structures of the 16S-23S ITS regions. Although, additional analyzes may be needed to answer this question (for example, house-keeping genes rpoB, gyrB etc., or ideally ANI comparison). But this is the maximum task. Although it is certainly extremely interesting, because we are talking about the concept of a species in cyanobacteria and the distribution areas of separate species (are there cosmoplite species?). Of course, within the framework of this manuscript, the authors may not solve this problem, but it would be interesting to mention and somehow discuss it.

We don't have a precise data for all samples of P. priestleyi, P. molle, and P. communis.

Reviewer 3 Report

The manuscript describes a new taxon in cyanobacteria. This is a very important aspect but the manuscript must be improved. The scientific writing is poor and rather confusing. I advice authors to read very carefully the manuscript.

Author Response

Dear reviewer,

We appreciate your time and productive suggestions which definitely improved the manuscript. We agree with most corrections, and we included them into the new version of the article. 

Round 2

Reviewer 1 Report

still require editing by a technical editor proficient in English

CHECK in text citation format; still unresolved

Table 1                check if the use of “Ibid” is appropriate; do you mean “same as above”???

lines 51-52        in text citation/check journal format in “The genus was described by Turicchia et al [15] by a comprehensive approach based on 16S rRNA gene phylogeny and defined as a separate genus phylogenetically allied to

Leptolyngbya s. st

15. Turicchia, S.; Ventura, S.; Komárková, J.; Komárek, J. Taxonomic evaluation of cyanobacterial microflora from alkaline marshesof northern Belize. 2. Diversity of oscillatorialean genera. Nova Hedwigia 2009, 89, 165–200. https://doi.org/10.1127/0029- 5705035/2009/0089-0165

line 75  sentence awkward; revise:         “Most parts of them Majority of the samples were collected by the senior author.”

line 76  remove dash “…collected by V. Redkina, #10 - by O. Rodina”

lines 76-77       “The Collected cyanobacterial samples were was taken and dried in a sterile paper bags then stored until needed for enrichment cultivation. Samples stored air-dry in the laboratory until enrichment cultivation uring.

line 106            “…characterized in previous ly studies [26-29].”

lines 110-113    “The data on the cyanobacterial diversity of the Polar Ural Mountains (Figure 1, #8, 9) and the main characteristics of sampling areas are given in a recent were previously reported in a previous study [30]. The main characteristics of sampling areas are were listed in cited articles.

“The cyanobacterial diversity of the Polar Ural Mountains (Figures 1, #8, 9) and the main characteristics of sampling areas were previously reported [30].

line 118            past tense          “…Cultures were are maintained…” 

line 228            ”… mingled with accessions based on Raabová et al. from [16] and other GB data…”

line 230            “…clade contained fifteen 15 Phormidesmis communis strains

line 235            “…obtained in by Raabová et al. [16] due to the transfer red of two accessions (KU219713 and KU219736) from PhP to PhN clade.

16. Raabová, L.; Kovacik, L.; Elster, J.; Strunecký, O. Review of the genus Phormidesmis (Cyanobacteria) based on environmental, morphological, and molecular data with description of a new genus Leptodesmis. Phytotaxa 2019, 395, 1–16. https://doi.org/10.11646/phytotaxa.395.1.1

lines 237-238    “The sequence analysis by eye allows to determine of three positions in 16S that could differentiate Phormidesmis priestleyi and P. nigrescens strains,,,,”

lines 245-248   “Raabová et al. The authors [16] tested four specimens named as Phormidesmis arctica that were placed in two sister-clades but with quite long branches and slight provided little support s and but authors suggested the samples them as a single species.”

            sentence not written technically; revise

lines 248-249    “The addition in the Current analysis of the members of the recently described ge-

nus, Alkalinema,  [3] entailed relation revealed relationship of the Phormidesmis arctica (PhA2)-clade to this genus…”

revise the sentence; sentence convoluted

comma before and after Alkalinema

line 252            delete colon      ”…were counted twice : ac-…”

line 258            spelling “which” in “…wich exceed…”

line 271            “…of the nearest relatives to ingroup taxa. – the genus Myxacorys – as is well-differentiated from…”

delete dash after “taxa” and Myxacorys; comma after the word “taxa” and Myxocorys

line 360            Clarify: form or from?   “Phormidesmis strains sequences deposited in GB database are not form the loner clade.”  Do you mean does not form a single clade?

line 272            in-text ref formatting again! “Following  Based on the studies by Yarza et al. , [45] the similarity…”

45. Yarza, P.; Yilmaz, P.; Pruesse, E.; Glöckner, L.W.; Schleifer, K.H.; Witman, W.B.; Euzéby, J.; Amann, R.; Rosselló-Móra Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nature Reviews Microbiology 2014, 12, 635–645. https://doi.org/10.1038/nrmicro3330

line 292   “Taking into account phylogenetic affinity and DNA sequence variability, …”

comma after variability

line 338   In-text ref formatting!           “In a publication Raabová et al. [16] failed to provide a holotype depository was not indicated for the Phormidesmis communis species”

16. Raabová, L.; Kovacik, L.; Elster, J.; Strunecký, O. Review of the genus Phormidesmis (Cyanobacteria) based on environmental, 572

morphological, and molecular data with description of a new genus Leptodesmis. Phytotaxa 2019, 395, 1–16. 573

https://doi.org/10.11646/phytotaxa.395.1.1

line 361   “The results suggested Phormidesmis has been shown to be a polyphyletic taxa.”

lines 361-362         revise the sentence; awkward              “The studying strains analyzed here form at least 4 four different lineages in the Phormidesmis and one clade in a sister position to Alkalinema, Myxacorys, Plectolyngbya andTapinothrix (Figure 2, 3).

usually no space before and after”/”; better use comma:   Alkalinema, Myxacorys, Plectolyngbya and Tapinothrix 

lines 365-366         “Several “Phormidesmis” strains are related to the different Leptolyngbyacae genera (Figure 2). 

Lines 371-373  “The reference s strain,  of Phormidesmis molle (Gomont), Turicchia, Ventura, Komárková  et Komárek [15] was characterized s as solitary or irregularly clustered filament, slightly coiled , and not attenuated at the ends [15].”

15. Turicchia, S.; Ventura, S.; Komárková, J.; Komárek, J. Taxonomic evaluation of cyanobacterial microflora from alkaline marshesof northern Belize. 2. Diversity of oscillatorialean genera. Nova Hedwigia 2009, 89, 165–200. https://doi.org/10.1127/0029-5035/2009/0089-0165 

Line 376              “End cells are rounded and do not differ from other vegetative cells, rounded at the end.”

lines 377-378    “Reference strain of Phormidesmis molle (3CC04S05), was isolated from alkaline marshes (Chan Chen) in northern Belize. The rRNA sequence of type the strain was not deposited in public data bases and cannot be used for comparison to compare.

line 380-383     “One of the strains Phormidesmis molle MMA66 (KR611710) bearing this name from

India falls into Phormidesmis communis clade. Another strain, s Phormidesmis molle, affiliated to with Leptolyngbya or different genera in Leptolyngbyaceae. Strain Phormidesmis molle PACC 8140 (KF770967) from Bulgaria doesn’t  does not belong to the Synechococcales.

Note:    no contraction in technical writing

lines 390-392   revise the sentence; sentence fragmented       “The article Taton et al. [46] adduce provided evidence that 9 nine different strains (ANT.L52.4, ANT.LG2.4, ANT.L52.6, ANT.LPR.5, ANT.LPR.6, ANT.L66.1, ANT.LMA.2, ANT.LACV5.1, ANT.L61.2) isolated from Cape Adare of James Ross Island, Antarctica, . which belong to the Phormidesmis priestley.”

line 393               delete “was”        “As was shown previously [16] strains…”

46. Taton, A.; Grubisic, S.; Ertz, D.; Hodgson, D.A.; Piccardi, R.; Biondi, N.; Tredici, M.R.; Mainini, M.; Losi, D.; Marinelli, F.; Wilmotte, A. Polyphasic study of Antarctic cyanobacterial strains. Journal of Phycology 2006, 42, 1257–1270. https://doi.org/10.1111/j.1529-8817.2006.00278.x

lines 395-397      “The o that taxon should be assign ed strains AB 12-2, AB 12-5, AB 12-6 from Antarctica, and G-49 from Sweden.

line 400            Should be plural; ”Some Phormidesmis strains…”;           ”… and confirmed that they it belong s to Phor…”

line 402                “Among Several strains in our collection follow a perfectly matched criteria set for…”

lines 406-407   “Macroscopically, mats have on agar with had a characteristic rusty color.

line 408            comma after ecosystems          “In Svalbard ecosystems, Phormidesmis…”

line 414                “…California (WJT36-NPBG12, WJT43-NPBG10) provided evidence that the

line 433            “The species is inhabits ing on wet rock and outcrops,…”

line 437            “Filaments with have thin colorless sheath…”

lines 439-440   “In cultures, cells are that forms wide ly and long cells (Figure 11b), rare trichomes are falsely pseudo-branched. Apical cells are rounded, and without calyptra.”

Lines 446-448  “This lineage may be considered as a loner the only genus in Leptolyngbyaceae, but formal ly relocated speciation es is not possible because the name of a the species is questionable. Invalidly published.”

Figure 9            remove dashes on the following:          (a) –       (b) – DIC              (c, d) – micro-photographs

lines 457-460      Specify the length and width          “The cell’s length is variable from shorter to up to twice longer than wide. From the studying samples, This was a single sample alone collected from the upper layer of anthropogenic podzol soil not in a subaerophytic habitat.”

line 461            “… are unreliable not reliably unable to distinguish the

species…”

line 467            “Follow by In agreement with other cyanobacterial taxonomists,

lines 474-476   “This is not a unique observation e, thus as observed in Myxacoris lineages were pointed to a likewise case [9]. The presence of two related taxa in nearby habitats suggests ing of

their niche differentiation.

line 484   “…subsequent molecular analyses allowed revealed s cryptic cyanobacteria diversity.

line 485            “Notwithstanding that Phormidesmis / Alkalinema / Apatinema species

Notwithstanding that Phormidesmis, Alkalinema and Apatinema species

Figure 11

remove dashes in the following:           (a) – habitat  (a) habitat

 (b-e) - DIC (b-e) DIC       (b) – strain (b) strain

(c) –KPABG (c) KPABG

(d) KPABGstill require editing by a technical editor proficient in English

CHECK in text citation format; still unresolved

Table 1                check if the use of “Ibid” is appropriate; do you mean “same as above”???

lines 51-52        in text citation/check journal format in “The genus was described by Turicchia et al [15] by a comprehensive approach based on 16S rRNA gene phylogeny and defined as a separate genus phylogenetically allied to

Leptolyngbya s. st

15. Turicchia, S.; Ventura, S.; Komárková, J.; Komárek, J. Taxonomic evaluation of cyanobacterial microflora from alkaline marshesof northern Belize. 2. Diversity of oscillatorialean genera. Nova Hedwigia 2009, 89, 165–200. https://doi.org/10.1127/0029- 5705035/2009/0089-0165

line 75  sentence awkward; revise:         “Most parts of them Majority of the samples were collected by the senior author.”

line 76  remove dash “…collected by V. Redkina, #10 - by O. Rodina”

lines 76-77       “The Collected cyanobacterial samples were was taken and dried in a sterile paper bags then stored until needed for enrichment cultivation. Samples stored air-dry in the laboratory until enrichment cultivation uring.

line 106            “…characterized in previous ly studies [26-29].”

lines 110-113    “The data on the cyanobacterial diversity of the Polar Ural Mountains (Figure 1, #8, 9) and the main characteristics of sampling areas are given in a recent were previously reported in a previous study [30]. The main characteristics of sampling areas are were listed in cited articles.

“The cyanobacterial diversity of the Polar Ural Mountains (Figures 1, #8, 9) and the main characteristics of sampling areas were previously reported [30].

line 118            past tense          “…Cultures were are maintained…”

line 228            ”… mingled with accessions based on Raabová et al. from [16] and other GB data…”

line 230            “…clade contained fifteen 15 Phormidesmis communis strains

line 235            “…obtained in by Raabová et al. [16] due to the transfer red of two accessions (KU219713 and KU219736) from PhP to PhN clade.

16. Raabová, L.; Kovacik, L.; Elster, J.; Strunecký, O. Review of the genus Phormidesmis (Cyanobacteria) based on environmental, morphological, and molecular data with description of a new genus Leptodesmis. Phytotaxa 2019, 395, 1–16. https://doi.org/10.11646/phytotaxa.395.1.1

lines 237-238    “The sequence analysis by eye allows to determine of three positions in 16S that could differentiate Phormidesmis priestleyi and P. nigrescens strains,,,,”

lines 245-248   “Raabová et al. The authors [16] tested four specimens named as Phormidesmis arctica that were placed in two sister-clades but with quite long branches and slight provided little support s and but authors suggested the samples them as a single species.”

            sentence not written technically; revise

lines 248-249    “The addition in the Current analysis of the members of the recently described ge-

nus, Alkalinema,  [3] entailed relation revealed relationship of the Phormidesmis arctica (PhA2)-clade to this genus…”

revise the sentence; sentence convoluted

comma before and after Alkalinema

line 252            delete colon      ”…were counted twice : ac-…”

line 258            spelling “which” in “…wich exceed…”

line 271            “…of the nearest relatives to ingroup taxa. – the genus Myxacorys – as is well-differentiated from…”

delete dash after “taxa” and Myxacorys; comma after the word “taxa” and Myxocorys

line 360            Clarify: form or from?   “Phormidesmis strains sequences deposited in GB database are not form the loner clade.”  Do you mean does not form a single clade?

line 272            in-text ref formatting again! “Following  Based on the studies by Yarza et al. , [45] the similarity…”

45. Yarza, P.; Yilmaz, P.; Pruesse, E.; Glöckner, L.W.; Schleifer, K.H.; Witman, W.B.; Euzéby, J.; Amann, R.; Rosselló-Móra Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nature Reviews Microbiology 2014, 12, 635–645. https://doi.org/10.1038/nrmicro3330

line 292   “Taking into account phylogenetic affinity and DNA sequence variability, …”

comma after variability

line 338   In-text ref formatting!           “In a publication Raabová et al. [16] failed to provide a holotype depository was not indicated for the Phormidesmis communis species”

16. Raabová, L.; Kovacik, L.; Elster, J.; Strunecký, O. Review of the genus Phormidesmis (Cyanobacteria) based on environmental, 572

morphological, and molecular data with description of a new genus Leptodesmis. Phytotaxa 2019, 395, 1–16. 573

https://doi.org/10.11646/phytotaxa.395.1.1

line 361   “The results suggested Phormidesmis has been shown to be a polyphyletic taxa.”

lines 361-362         revise the sentence; awkward              “The studying strains analyzed here form at least 4 four different lineages in the Phormidesmis and one clade in a sister position to Alkalinema / Myxacorys / Plectolyngbya / Tapinothrix (Figure 2, 3).

usually no space before and after”/”; better use comma:   Alkalinema, Myxacorys, Plectolyngbya and Tapinothrix

lines 365-366         “Several “Phormidesmis” strains are related to the different Leptolyngbyacae genera (Figure 2).

Lines 371-373  “The reference s strain,  of Phormidesmis molle (Gomont), Turicchia, Ventura, Komárková  et Komárek [15] was characterized s as solitary or irregularly clustered filament, slightly coiled , and not attenuated at the ends [15].”

15. Turicchia, S.; Ventura, S.; Komárková, J.; Komárek, J. Taxonomic evaluation of cyanobacterial microflora from alkaline marshesof northern Belize. 2. Diversity of oscillatorialean genera. Nova Hedwigia 2009, 89, 165–200. https://doi.org/10.1127/0029-5035/2009/0089-0165

Line 376              “End cells are rounded and do not differ from other vegetative cells, rounded at the end.”

lines 377-378    “Reference strain of Phormidesmis molle (3CC04S05), was isolated from alkaline marshes (Chan Chen) in northern Belize. The rRNA sequence of type the strain was not deposited in public data bases and cannot be used for comparison to compare.

line 380-383     “One of the strains Phormidesmis molle MMA66 (KR611710) bearing this name from

India falls into Phormidesmis communis clade. Another strain, s Phormidesmis molle, affiliated to with Leptolyngbya or different genera in Leptolyngbyaceae. Strain Phormidesmis molle PACC 8140 (KF770967) from Bulgaria doesn’t  does not belong to the Synechococcales.

Note:    no contraction in technical writing

lines 390-392   revise the sentence; sentence fragmented       “The article Taton et al. [46] adduce provided evidence that 9 nine different strains (ANT.L52.4, ANT.LG2.4, ANT.L52.6, ANT.LPR.5, ANT.LPR.6, ANT.L66.1, ANT.LMA.2, ANT.LACV5.1, ANT.L61.2) isolated from Cape Adare of James Ross Island, Antarctica, . which belong to the Phormidesmis priestley.”

line 393               delete “was”        “As was shown previously [16] strains…”

46. Taton, A.; Grubisic, S.; Ertz, D.; Hodgson, D.A.; Piccardi, R.; Biondi, N.; Tredici, M.R.; Mainini, M.; Losi, D.; Marinelli, F.; Wilmotte, A. Polyphasic study of Antarctic cyanobacterial strains. Journal of Phycology 2006, 42, 1257–1270. https://doi.org/10.1111/j.1529-8817.2006.00278.x

lines 395-397      “The o that taxon should be assign ed strains AB 12-2, AB 12-5, AB 12-6 from Antarctica, and G-49 from Sweden.

line 400            Should be plural; ”Some Phormidesmis strains…”;           ”… and confirmed that they it belong s to Phor…”

line 402                “Among Several strains in our collection follow a perfectly matched criteria set for…”

lines 406-407   “Macroscopically, mats have on agar with had a characteristic rusty color.

line 408            comma after ecosystems          “In Svalbard ecosystems, Phormidesmis…”

line 414                “…California (WJT36-NPBG12, WJT43-NPBG10) provided evidence that the

line 433            “The species is inhabits ing on wet rock and outcrops,…”

line 437            “Filaments with have thin colorless sheath…”

lines 439-440   “In cultures, cells are that forms wide ly and long cells (Figure 11b), rare trichomes are falsely pseudo-branched. Apical cells are rounded, and without calyptra.”

Lines 446-448  “This lineage may be considered as a loner the only genus in Leptolyngbyaceae, but formal ly relocated speciation es is not possible because the name of a the species is questionable. Invalidly published.”

Figure 9            remove dashes on the following:          (a) –       (b) – DIC              (c, d) – micro-photographs

lines 457-460      Specify the length and width          “The cell’s length is variable from shorter to up to twice longer than wide. From the studying samples, This was a single sample alone collected from the upper layer of anthropogenic podzol soil not in a subaerophytic habitat.”

line 461            “… are unreliable not reliably unable to distinguish the

species…”

line 467            “Follow by In agreement with other cyanobacterial taxonomists,

lines 474-476   “This is not a unique observation e, thus as observed in Myxacoris lineages were pointed to a likewise case [9]. The presence of two related taxa in nearby habitats suggests ing of

their niche differentiation.

line 484   “…subsequent molecular analyses allowed revealed s cryptic cyanobacteria diversity.

line 485            “Notwithstanding that Phormidesmis / Alkalinema / Apatinema species

Notwithstanding that Phormidesmis, Alkalinema and Apatinema species

Figure 11

remove dashes in the following:           (a) – habitat  (a) habitat

 (b-e) - DIC (b-e) DIC       (b) – strain (b) strain

(c) –KPABG (c) KPABG

(d) KPABG

(e) KPABG-125316.

(e) KPABG-125316.

Author Response

Dear reviewer, Thanks a lot for your great editing ogf our MS! We appreciate your time and productive suggestions which definitely improved Phormidesmis manuscript. We agree with corrections, and we included them into the new version of the article.

Reviewer 2 Report

Dear Authors,

You have made some changes to the article and gave your answers on my comments, but I am not completely satisfied with it.

You disregarded my recommendation to clearly compare the sequences of your strains with the type/reference strains of the Phormidesmis species.

Figs 2 and 3: The emphasis in bold for non-Phormidesmis strains isolated by the authors is still confusing. The meaning of bolding them is not clear. I still think that the presented phylogenetic trees are redundant and the number of non-Phormidesmis strains can be reduced. But I accept the explanation of the authors (although it does not contain clear arguments) and remove this remark.

The authors added 16S-23S ITS secondary structures but its discussion is highly unsatisfactory. Discussion of these results in the context of the differences between the genera Phormidesmis and Apatinema is of little importance, since these genera are perfectly distinguished by the 16S criterion. But in order to deal with the intrageneric differences of Phormidesmis strains, these data are needed. Preliminarily, quite significant differences between the strains are visible, which may indicate the possibility of some strains to be a separate species. This should be analyzed in more depth and discussed in the article.

Still, a description of the morphology and the habitats of the strains isolated and identified by the authors should be removed to the Results section.  Generalizations for all known strains based on these data and including literature data should be made in the Discussion section.

Additionally:
1. Please, correct Phormisesmis to Phormidesmis throughout the text, including phylogenetic trees. Much has already been fixed, but far from everything.

2. Please, correct Tables 1, 3. It will be nice if you add the taxonomic names of the isolates to it, and not just collection IDs of the strains.

3. Please, uniform way of writing of collection numbers everywhere throughout the text including the phylogenetic trees. In the present manuscript the full names of the strains are indicated (for example, KPABG 3629) as well as only numbers (3629). This creates some difficulty while reading.

4. Please, correct Table 2: indicate that "P. arctica 2" corresponds to Apatinema mutabilis.

Dear Authors! In general, I support you. The manuscript seems interesting to me and worthy of publication. But as an outside reader, I see that it is rather complicated and inconsistently written in the present state. I think the manuscript requires optimization of the text and  material presentation. I hope that careful attention to my comments will improve the manuscript and make it more attractive to the scientific community.

Author Response

Dear Reviewer! Thank you for your support! We tried to improve the MS as you recommend.

Dear Reviewer! Thank you for your support! We tried to improve the MS as you recommend. 
We added the analysis of the secondary structures in Results and Discussion. Obviously, we observe here the great nucleotide sequence diversity in the ITS region between natural populations. We could consider each strain as a loner species but, perhaps, they appear to be a member of a widely distributed variable species.

Reviewer 3 Report

The changes made in the manuscript were very usefull to clarify it.

Author Response

Thanks for your effort!

Round 3

Reviewer 2 Report

Dear Authors!

This time you have done an excellent job and I am satisfied with the results. In my opinion, the article has become much better and acquired scientific significance. But please check your English carefully! There are still many mistakes.