A New Deep-Water Epilithic Green Alga, Ulvella lacustris, from an Alpine Brackish Lake in Qinghai–Tibet Plateau

Round 1

Reviewer 1 Report

The manuscript presents a new species of the genus Ulvella found on rocks from the deep zone of an alpine brackish lake in China. The paper is very well presented and the authors deserve congratulations.

There are only a few small details that should be corrected for the final version.

First, in the heading of Table 1, the meaning of the abbreviations used should be indicated and pH should be spelled correctly.

Secondly, they should revise the whole manuscript and write in italic font the names of the taxa as usual; in some places they appear without italics, for example in the caption of Figure 2, Figure 3, Figure 20.

With respect to figures 19 and 20, as they are placed side by side, it would be more appropriate for figure 19 to be 19a and figure 20 to be 19b, so that the same caption would serve for both figures. As it is now, it is not adequate.

Finally, just revise the list of bibliographic references so that all of them are in the style of the journal. For example, in some of them the texts appear in all capital letters.

Author Response

Dear Editors and Reviewers:

Thank you to the reviewers for your valuable suggestions on the manuscript during your busy schedule. We have made corresponding revisions to the problems in the article, including polishing and standardizing the language and format. The specific revisions are as follows:

The manuscript presents a new species of the genus Ulvella found on rocks from the deep zone of an alpine brackish lake in China. The paper is very well presented and the authors deserve congratulations.

Re：Thank you for your affirmation and compliment !

First, in the heading of Table 1, the meaning of the abbreviations used should be indicated and pH should be spelled correctly.

Re：Fixed.

Secondly, they should revise the whole manuscript and write in italic font the names of the taxa as usual; in some places they appear without italics, for example in the caption of Figure 2, Figure 3, Figure 20.

Re：Fixed

With respect to figures 19 and 20, as they are placed side by side, it would be more appropriate for figure 19 to be 19a and figure 20 to be 19b, so that the same caption would serve for both figures. As it is now, it is not adequate.

Re：Fixed

Finally, just revise the list of bibliographic references so that all of them are in the style of the journal. For example, in some of them the texts appear in all capital letters.

Re：Fixed.

Reviewer 2 Report

Review of “A new free-living epilithic green alga, Ulvella lacustris, from 2 abyssalzone of alpine brackish lake in Qinghai-Tibetan Plateau” by Qiufeng Yan et al. for the journal Diversity

The English needs considerable editing. I give just an example with the title and the abstract. Here edited. I did not edit the remainder of the text but suggest having a native English speaker check the manuscript.

A new free-living epilithic green alga, Ulvella lacustris, from the abyssal zone of alpine brackish lake in Qinghai-Tibetan Plateau

Abstract. Ulvella species are widely distributed in marine and freshwater habitats and from high latitudes to the tropics. However, no species of this genus has been found in the Qinghai-Tibetan Plateau. In the present study, three unialgal strains were isolated from rocks collected from the bottom of Qinghai Lake at a depth between 15 and 28 m. These strains exhibited prostrate pseudoparenchyma, irregular or radial branching filaments, and mono- or polystromatic discs or rosettes formed by consecutive filaments. Thalli grow up to 180 um in length. Phylogenetic trees inferred from 18S rDNA and tufA cpDNA sequences revealed that the isolates were resolved among other Ulvella species and were closely related to Ulvella leptochaete and Ulvella waernii. This result was also confirmed by a neighbor joining tree inferred from ITS2 aligned using a secondary structure model. Unlike other members of Ulvella, the isolated strains were characterized by a unique habitat, a distinct field morphology of the thallus, size of the vegetative cells, and number of pyrenoids. Based on these morphological differences, phylogenetic analysis, and comparison of ITS2 secondary structure with related species, the strains isolated in this study are proposed to belong to a species new to science, Ulvella lacustris sp. nov.

30 What are ‘primary plants’?

58 thallus singular; thalli plural

79 here is mentioned 5 specimens, in abstract 3

80 ships and other lost valuables at sea are salvaged; rocks and plants are collected

84 speices?

111 mention primer concentration and what is in the mastermix

122 which genbank numbers refer to which sequences?

130 TAMUER and NEI (1993) and KIMUER (1980) not conform referencing. Moreover, Names look misspelled. Maybe Kimura and Tamura and Nei?

125-146 phylogenetic analysis is well-done

151 ClustalW?

172 A formal description needs to mention also where the type material can be accessed, where a strain of the type specimen is accessible, where the exact type locality is. And which GenBank sequences refer to this type specimen. You can then also designate the other strains as isolectotypes. All this can be done at the end of theresults.

192 Figure 9 should read Figures 9-16. Illustrations are clear and of good quality.

200 before 3.2 Phylogenetic analysis I suggest first describing the obtained sequences from the strains. A simple table with strains, the genbank numbers of sequences for 18S, Tuf A and ITS

220 In tree Ulvella lacustris sp. nov. Since the interest is here on the phylogenetic position of the Ulvella strains I strongly advise to remove Chlorophyceae and Trebouxiophyceae and choose Oltmann-Scotino-, Ignat- as outgroup. That gives a nice nested set of ingroups and is more than enough. This paper is not about the phylogeny of the green plants as a whole.

224: for tufa tree, were there no TufA from Ulva, Percursaria, Ulvaria, Umbraulva and other close outgroups? I would remove all PP-values below 0.95 from the tree and replace with ‘-‘ as such values are not significant

231-242 It is not clear to me what the ITS2 secondary structure analysis adds to the study. Yes, the strains’ ITS2 differ from those of four other species, but then so what? 

242 Numbering of the figures and figure legends do not match. Fig. 20 In order to make the information in this figure reproducible by others we need to know the genbank numbers of the sequences used. Why only one ITS sequence of U. lacustris? Are these the ones metioned in Table 3. State so!

252 Why is the heading of the table not aligned?

254-266 is not relevant to the results of the present study and needs to be eliminated. This is all review of earlier work to which U. lacustris does not add. Discussion should focus on the placement of U. lacustris. If there is anything in this species that challenges the current classification of the Ulvophyceae based on morphological characteristics, then that is worth discussion. Otherwise, do not expand into the classification of green plants. 

274-285 CBC correlate somehow with species barriers but are not indicative. The citations 46-48 are somewhat antique. Do the authors know of recent literature in which CBCs are used to separate species? 

290 what is a unique phylogeny?

300 habitat difference refers to rocks or reed, or to ‘near the surface versus 15-20 m deep

318 Known things can be neglected, unknown things need to be discovered. You note that these high altitude brackish lakes may contain quite an interesting diversity and may need to be explored better to uncover the true extent of their diversity before economic development destroys the biodiversity of these habitats. 

Citations are to the point and figures are clear.

Author Response

30 What are ‘primary plants’?

Re：lower plants.( We have deleted the first paragraph)

58 thallus singular; thalli plural

Re：Fixed.

79 here is mentioned 5 specimens, in abstract 3

Re：Fixed.(Three strains of algae were isolated from five samples.)

80 ships and other lost valuables at sea are salvaged; rocks and plants are collected

Re：Fixed.

84 speices?

Re：Fixed.

111 mention primer concentration and what is in the mastermix

Re：Fixed.

122 which genbank numbers refer to which sequences?

Re：The correspondence between genbank numbers and sequences is shown in the evolutionary tree.

130 TAMUER and NEI (1993) and KIMUER (1980) not conform referencing. Moreover, Names look misspelled. Maybe Kimura and Tamura and Nei?

Re：Corrected spelling and references.

125-146 phylogenetic analysis is well-done

Re：Thank you for your recognition.

151 ClustalW?

Re：A tool for 4SALE.

172 A formal description needs to mention also where the type material can be accessed, where a strain of the type specimen is accessible, where the exact type locality is. And which GenBank sequences refer to this type specimen. You can then also designate the other strains as isolectotypes. All this can be done at the end of theresults.

Re：The distribution of this species is mentioned later, and there are GenBank sequences in the phylogenetic tree.

192 Figure 9 should read Figures 9-16. Illustrations are clear and of good quality.

Re：Thank you.

200 before 3.2 Phylogenetic analysis I suggest first describing the obtained sequences from the strains. A simple table with strains, the genbank numbers of sequences for 18S, Tuf A and ITS

Re：The genbank numbers are already shown in the phylogenetic tree, so no separate table is prepared.

220 In tree Ulvella lacustris sp. nov. Since the interest is here on the phylogenetic position of the Ulvella strains I strongly advise to remove Chlorophyceae and Trebouxiophyceae and choose Oltmann-Scotino-, Ignat- as outgroup. That gives a nice nested set of ingroups and is more than enough. This paper is not about the phylogeny of the green plants as a whole.

Re：I very much agree with your point of view. Since we did not know what algae this was at first, we used the Chlorophyceae and Trebouxiophyceae taxa to construct the evolutionary tree, and this process also reflected our knowledge of the phylogenetic position of this alga, which also allowed us to confirm the algae belong to Ulvophyceae, and we only involved the group of Ulvophyceae in the phylogenetic tree constructed by TufA to determine the phylogenetic position within the genus Ulvella.

224: for tufa tree, were there no TufA from Ulva, Percursaria, Ulvaria, Umbraulva and other close outgroups? I would remove all PP-values below 0.95 from the tree and replace with ‘-‘ as such values are not significant

Re：Thanks for your advice.TufA is to explore the interspecific relationship of Ulvella, so only three extra-algal taxa were used.

231-242 It is not clear to me what the ITS2 secondary structure analysis adds to the study. Yes, the strains’ ITS2 differ from those of four other species, but then so what? 

Re：In previous studies, the emergence of CBCs is often used as an indicator of isolated species or genera, and this difference may make our new species more convincing.

242 Numbering of the figures and figure legends do not match. Fig. 20 In order to make the information in this figure reproducible by others we need to know the genbank numbers of the sequences used. Why only one ITS sequence of U. lacustris? Are these the ones metioned in Table 3. State so!

Re：Figure numbering has been corrected. The genbank numbers are listed in the tree. The ITS sequences of the three algae are completely identical, so only one is used here.

252 Why is the heading of the table not aligned?

Re：Fixed.

254-266 is not relevant to the results of the present study and needs to be eliminated. This is all review of earlier work to which U. lacustris does not add. Discussion should focus on the placement of U. lacustris. If there is anything in this species that challenges the current classification of the Ulvophyceae based on morphological characteristics, then that is worth discussion. Otherwise, do not expand into the classification of green plants. 

Re：254-266 has been eliminated.

274-285 CBC correlate somehow with species barriers but are not indicative. The citations 46-48 are somewhat antique. Do the authors know of recent literature in which CBCs are used to separate species? 

Re：CBC is not indicative and serves as a reference here.

290 what is a unique phylogeny?

Re：Fixed.

300 habitat difference refers to rocks or reed, or to ‘near the surface versus 15-20 m deep

Re：Fixed.

318 Known things can be neglected, unknown things need to be discovered. You note that these high altitude brackish lakes may contain quite an interesting diversity and may need to be explored better to uncover the true extent of their diversity before economic development destroys the biodiversity of these habitats. 

Re：Fixed.

Reviewer 3 Report

Dear Authors,

Is the manuscript clear, relevant for the field and presented in a well-structured manner?

I think that the study is valuable and relevant to the field. In general, the article is mostly clearly presented and the structure makes sense to me.

Having written this, I'd also like to mention the some difficulties I encountered. 

Specific comments:

Line 11: “Ulvella”. Remove the bold.

Line 30-47: The Chlorophyta group has been too broadly described!

Line 72-79: Please, introduce more information about habitat, distribution, biology and ecology of study species.

Line 183-4: “..and the cell wall and integument were thicker (Figs 4-5)”. The photos do not show the thickness of the cell wall. Authors should take photos with TEM and SEM.

Please, read the paper by Bartolo, A.G., Zammit, G., Küpper, F.C. Germling culture and molecular analysis of evasive microfilamentous green algae growing in the Maltese islands (central Mediterranean). Botanica Marina, 2022.

Please, write the introduction again.

Line 174: should be Figures 2-8. Whether the Authors have better quality photos? I can’t see any taxonomic (morphological) details of Ulvella.

Line 192: should be Figures 9-16. Please, introduce the arrows for chloroplasts and pyrenoids. I am confused (see Line 195).

Comments for author File: Comments.pdf

Author Response

Line 11: “Ulvella”. Remove the bold.

Re：Fixed.

Line 30-47: The Chlorophyta group has been too broadly described!

Re：Thanks for your advice. We've made adjustments to the first paragraph.

Line 72-79: Please, introduce more information about habitat, distribution, biology and ecology of study species.

Re：Habitat, distribution, etc. are described later.

Line 183-4: “..and the cell wall and integument were thicker (Figs 4-5)”. The photos do not show the thickness of the cell wall. Authors should take photos with TEM and SEM.

Re：The concept of cell wall thickness is indeed ambiguous here, and the description has been Fixed.

Please, read the paper by Bartolo, A.G., Zammit, G., Küpper, F.C. Germling culture and molecular analysis of evasive microfilamentous green algae growing in the Maltese islands (central Mediterranean). Botanica Marina, 2022.

Re：OK, I'll take your suggestion into consideration.

Please, write the introduction again.

Re：Some modifications have been made.

Line 174: should be Figures 2-8. Whether the Authors have better quality photos? I can’t see any taxonomic (morphological) details of Ulvella.

Re：Sorry, we tried to retake the photo, but it wasn't as good as it is now.

Line 192: should be Figures 9-16. Please, introduce the arrows for chloroplasts and pyrenoids. I am confused (see Line 195).

Re：This is an erroneous description, which has been corrected in the manuscript.

Round 2

Reviewer 2 Report

Review of Yan et al. A new free-living epilithic green alga, Ulvella lacustris, from abyssal zone of alpine brackish lake in Qinghai-Tibetan Plateau.

The title needs fixing. I suggest: A new deep-water epilithic green alga, Ulvella lacustris, from an alpine brackish lake in Qinghai-Tibetan Plateau. Note that the definition of Abyssal denotes the depths or bottoms of the ocean, especially between about 3000 and 6000 meters down or from 2000 to 5000 meters down depending on the reference. So 20 m depth in a Tibetan lake is not abyssal. There is also a problem with free-living epilithic because free-living means “living freely and independently, not as a parasite or attached to a substrate” and epilithic means “growing on the surface of rock.” So, what is the habitat?

Line 9: The first sentence in the abstract now reads “Species are widely distributed in the ocean and freshwater habitats and from high latitudes to tropics”, which is basically an empty statement.

Lines 78-80: How can one collect three algal specimens from five different rocks?

Fig. 1. Since there is a legend in this figure, the statement in the figure legend, Green represents the Ulvella sample found is not only grammatically incorrect but superfluous. To give the reader a clue I would print the QHH sample numbers next to the green dots. I would put the altitude of the lake surface (together with the name of the lake) in the figure legend, and a km scale bar in the figure.

In Table 1 I assume the Altitude refers to the lake surface; please could you explain how it can be meters different at different sites? In the Table I would list the sample depths in the place of the Altitude.

Line 112-113: 10mol/L of each primer cannot be correct. One cannot dissolve so many kg of primer in a liter of water. Concentrations and brand of Polymerase and PCR buffer and concentrations of dNTP in the reaction mix must be provided. Please write the correct information, rather than just something.

In my comments to version 1 I wrote “172 A formal description needs to mention also where the type material can be accessed, where a strain of the type specimen is accessible, where the exact type locality is. And which GenBank sequences refer to this type specimen. You can then also designate the other strains as isolectotypes. All this can be done at the end of the results.” The answer: “The distribution of this species is mentioned later, and there are GenBank sequences in the phylogenetic tree” Does not address the issue. If the authors wish to validly describe the species, they need to abide by the international nomenclatural rules.

In my comments to version 1 I wrote 220 In tree [Fig 17] Ulvella lacustris sp. nov. Since the interest is here on the phylogenetic position of the Ulvella strains I strongly advise to remove Chlorophyceae and Trebouxiophyceae and choose Oltmann-Scotino-, Ignat- as outgroup. That gives a nice nested set of ingroups and is more than enough. This paper is not about the phylogeny of the green plants as a whole.

Re: I very much agree with your point of view. [Good.] Since we did not know what algae this was at first, we used the Chlorophyceae and Trebouxiophyceae taxa to construct the evolutionary tree, and this process also reflected our knowledge of the phylogenetic position of this alga, which also allowed us to confirm the algae belong to Ulvophyceae, and we only involved the group of Ulvophyceae in the phylogenetic tree constructed by TufA to determine the phylogenetic position within the genus Ulvella.

The answer is contradictory: “I very much agree with your point of view.“ But I don’t follow the advice. In the way of scientific process of exploration, “Where does this species belong?” can be answered with a simple BLAST search. Answer: “Oh, In the Ulvellaceae!” Then the phylogenetic tree construction can be done with the Ulvellaceae and with the other Ulvophyceae as nested set of outgroups. I notice that there is a clade with Oltmannsiellopsidaceae and another one with Oltmansiellopsidales? Why different taxonomic levels (ceae and ales) in the same tree?

In my comment to version 1 I wrote: 224: for tufa tree, were there no TufA from Ulva, Percursaria, Ulvaria, Umbraulva and other close outgroups? [AND] I would remove all PP-values below 0.95 from the tree and replace with ‘-‘ as such values are not significant

"Re: Thanks for your advice. TufA is to explore the interspecific relationship of Ulvella, so only three extra-algal taxa were used."

Answers the first part of the query. The second part of the query remains unaddressed because I still see PP-values below 0.95 in Fig. 18

Line 226 Fig. 18 is a tufa tree according to the figure legend. In the Discussion we read in Line 280-281 “This finding was confirmed by the neighbor-joinING tree based on ITS2 secondary structure (Fig. 18).”

Note that Discussion is normally written in present tense because it discusses the results found rather that repeating them.

Line 301: what is a clear phylogeny of U. lacustris’ A clear distinction of U. lacustris from other species. To me, the tufA sequence distinction between U. lacustris and U. waernii is not at all clear

In my comment 300 habitat difference refers to rocks or reed, or to ‘near the surface versus 15-20 m deep? Re：Fixed. My response. It has not been fixed by eliminating “difference” in line 310. What habitat? I suggest, “These habitat differenceS may explain the morphological differences between….

I do not know if the Authors have information about the morphology of all the strains from which they included GenBank sequences in their tree, but it might be interesting to do a formal, more in-depth, analysis of the changes in morphological characters all over the tufA tree. Which characters change state all over the place and which ones change state only once or a few times. The latter are probably stable and phylogenetically informative whereas the former could result merely from morphological plasticity.  Why do that? Well, if the differences between U. waernii and U. lacustris are all in the former category, then that suggests geographically distant specimens belonging to the same species because the genetic differences are slight. Info about U waernii is in ref. 18. All this can be reflected against information about characters that change state when culture conditions change or when material is brought into culture. Note also that tufA sequence differences between different geographic isolates of the same species are common in the tree in Fig. 18

I basically suspect that the strains of U. lacustris are just geographically distinct isolates of U. waernii. ... Unless you can make clear that the morphological differences between the two are stable features that do not change as a result of environmental differences. The discussion meanders over these issues without coming to a clear conclusion on the matter. A more formal analysis of these morphological differences is therefore due  

Author Response

The title needs fixing. I suggest: A new deep-water epilithic green alga, Ulvella lacustris, from an alpine brackish lake in Qinghai-Tibetan Plateau. Note that the definition of Abyssal denotes the depths or bottoms of the ocean, especially between about 3000 and 6000 meters down or from 2000 to 5000 meters down depending on the reference. So 20 m depth in a Tibetan lake is not abyssal. There is also a problem with free-living epilithic because free-living means “living freely and independently, not as a parasite or attached to a substrate” and epilithic means “growing on the surface of rock.” So, what is the habitat?

Re：Fixed

Line 9: The first sentence in the abstract now reads “Species are widely distributed in the ocean and freshwater habitats and from high latitudes to tropics”, which is basically an empty statement.

Re：Fixed

Lines 78-80: How can one collect three algal specimens from five different rocks?

Re：Algae isolated from the other two samples were not successfully cultured.

Fig. 1. Since there is a legend in this figure, the statement in the figure legend, Green represents the Ulvella sample found is not only grammatically incorrect but superfluous. To give the reader a clue I would print the QHH sample numbers next to the green dots. I would put the altitude of the lake surface (together with the name of the lake) in the figure legend, and a km scale bar in the figure.

Re：Fixed

In Table 1 I assume the Altitude refers to the lake surface; please could you explain how it can be meters different at different sites? In the Table I would list the sample depths in the place of the Altitude.

Re：Both sample depths and Altitude are listed in Table 1.

Line 301: what is a clear phylogeny of U. lacustris’ A clear distinction of U. lacustris from other species. To me, the tufA sequence distinction between U. lacustris and U. waernii is not at all clear

 Re：Fixed