Hoefkenia hunsrueckensis, a New Genus and Species from Europe, and the Identity of Virescentia vogesiaca (F.W.Schultz ex Skuja) Necchi, D.C.Agostinho & M.L.Vis (Batrachospermales, Rhodophyta)
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript has serious flaws and problems. First, the general aim is weak: showing that V. vogesiaca identified from Germany does not belong to that species is only of regional interest. Secondly, the authors proposed a new genus to accommodate that alga based on unreliable and poorly presented dataset; the proposal is clearly a big mistake since the alga was not compared with the closest related taxa from Europe, particularly those of the genus Kumanoa. Third, the phylogenetic analyses based on DNA sequences were not properly conducted/presented: a) a rectangular cladogram is not acceptable nowadays and should be replaced by a phylogram to show the branch lengths; b) no sequence divergence values among the sequences of the study alga with the closest taxa are given to make it clear the difference/similarity with the closest related taxa.
The clade with the study samples are closely related to one with three species of Kumanoa, which is the most species rich genus in the Batrachospermales with around 40 species. Thus, additional species have to be included in the analyses, particularly those occurring in Europe (K. abilii, K. globospora, K. mahlacensis and K. virgatodecaisneana) but other species as well. A preliminary sequence comparison with known species should be done with BLAST. Based on the molecular data and the morphology, I have reasons to believe that the study alga is a species of Kumanoa, perhaps not even a new species.
Thus, my recommendation is to reject the manuscript in the present content and format. Authors should completely rewrite the manuscript under a different perspective (focusing on the comparison with Kumanoa species) before resubmitting it. If the new results prove that the alga is a new species, the manuscript could be submitted to this journal but if not a local journal showing the misidentification would be suitable.
Comments on the Quality of English LanguageQuality of English is OK, the content is the most problematic issue.
Author Response
The manuscript has serious flaws and problems. First, the general aim is weak: showing that V. vogesiaca identified from Germany does not belong to that species is only of regional interest.
Response: This is not the general aim. The paper helps to clarify taxonomic errors and confusion found in the literature and provides significant evidence for an overlooked species that is genetically and morphologically highly distinct and has no affinities to the species Virescentia vogesiaca under which it is currently recorded. Besides reporting a truly new plant species or genus that thrives in such a pure ecosystem is quite something for the European flora.
Secondly, the authors proposed a new genus to accommodate that alga based on unreliable and poorly presented dataset; the proposal is clearly a big mistake since the alga was not compared with the closest related taxa from Europe, particularly those of the genus Kumanoa.
Response: The latest complex analysis of Kumanoa was published in Plant and Fungal Systematics (Fischer at al. 2020. PFS 65:147-166) where the breath of the genus is fully analysed in conjunction with other previous analyses (see below). In the current analyses we reduced the data set presented in PFS 2020 to the core species of the different Kumanoa clades as the generic evolution of Kumanoa is not in the focus of this paper and has already been presented (see below). European Kumanoa species are deeply nested inside Kumanoa, and are thus not relevant at this stage (compare Vis at al. 2012: Molecular phylogeny of the genus Kumanoa (Batrachospermales, Rhodophyta) and Fischer at al. 2020), especially as Kumanoa itself is a maximally supported monophylum by Baysian inference, maximum likelihood and parsominy
Third, the phylogenetic analyses based on DNA sequences were not properly conducted/presented: a) a rectangular cladogram is not acceptable nowadays and should be replaced by a phylogram to show the branch lengths; b) no sequence divergence values among the sequences of the study alga with the closest taxa are given to make it clear the difference/similarity with the closest related taxa.
Response: Considering the statement that the phylogenetic analyses based on DNA sequences were not properly conducted/presented is highly irritating, as state-of-the-art (BI. ML) methods were used for the phylogenetic inferences and clade support estimates. The notion that a phylogram should be presented is puzzling as it is provided in the figure. The rectangular version of the tree was choosen in order to properly visualize the important clade support and the phylogram is depicted exactly to show the length of the branches separating Kumanoa and the new genus Hoefkenia.
The clade with the study samples are closely related to one with three species of Kumanoa, which is the most species rich genus in the Batrachospermales with around 40 species. Thus, additional species have to be included in the analyses, particularly those occurring in Europe (K. abilii, K. globospora, K. mahlacensis and K. virgatodecaisneana) but other species as well. A preliminary sequence comparison with known species should be done with BLAST.
Response: As outlined above a comprehensive phylogeny of Kumanoa has been published by Vis et al. (2012) that we extended almost reaching taxonomic completion of the genus (Fischer et al. 2020). Here we have included representatives of all clades within the genus according to Vis et al. (2012) and Fischer et al. (2020). For Virescentia and Paludicola we have enlarged the samplimg adding all available species including our own collections. The outlined species by the reviewer Kumanoa globospora and K. virgatodecaisnea were already included in the analyses as representatives of a derived Kumanoa clade. As to K. abilii, Vis et al. (2012), Necchi et al. (2010) and Fischer at al. (2020) have already shown that this species is part of the K. rwandensis / K. ambigua clade (see below) which we represented by K. rwandensis. Thus, this taxon is deep inside Kumanoa far away from the new taxon. Similarly, K. mahlacensis is deeply nested inside Kumanoa and thus of no concern. As commented above, a generic inclusion of the new taxon in Kumanoa would render other genera like Virescentia, Paludicola and Visia as synonyms of Kumanoa The K. rwandensis / K. ambigua clade and the position of K. abilii (Fischer et al. 2020. Plant and Fungal Systematics (65: 147–166, 2020)
Based on the molecular data and the morphology, I have reasons to believe that the study alga is a species of Kumanoa, perhaps not even a new species.
Response: All European Kumanoa species are inside the maximally supported monophyletic genus Kumanoa. Hoefkenia hunsreckensis is sister to Kumanoa and genetically and morphologically very distinct from Kumanoa which supports the decision of erecting a genus for the ignored taxon. The main distinguishing character of Kumanoa is the twisted or spirally coiled carpogonial branch while that of Virescentia and the new taxon is straight. Thus, the new taxon does not share the derived characters of Kumanoa. Table 3 gives an overview of the morphological differences between the new Hoefkenia and Kumanoa, Virescentia, Visia and Paludicola. It shows that the morphological differences are often not very marked
Thus, my recommendation is to reject the manuscript in the present content and format. Authors should completely rewrite the manuscript under a different perspective (focusing on the comparison with Kumanoa species) before resubmitting it. If the new results prove that the alga is a new species, the manuscript could be submitted to this journal but if not a local journal showing the misidentification would be suitable.
Response: There is no need to rewrite the manuscript as all results and conclusions are clearly explained and significantly show that it is a new species. The focus of the comparison with Kumanoa is already made.
Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors
The study that you present is interesting and novel in the knowledge of freshwater Rhodophyta.
I suggest you review the following:
In methodology, indicate why the external group was chosen. The selection of the two markers is because it is recommended in the bibliography.
It is necessary to correct the legends of figures and add its forgot about them.
The most important thing is to support in a more robust way the morphological differences between species and genera.
Other observations are highlighted in the manuscript
Comments for author File: 
Comments.pdf
Author Response
The study that you present is interesting and novel in the knowledge of freshwater Rhodophyta.
I suggest you review the following:
In methodology, indicate why the external group was chosen. The selection of the two markers is because it is recommended in the bibliography.
Response: The chose of the outgroup should reflect other European freshwater red-algae that do not belong tot he Batrachospermales and Batrachospermaceae. It has been proven to be effective as it was already used in a study of African freshwater red algae (Fischer et al., Plant and Fungal Systematics (65(1): 147–166, 2020). The two markers are those used in nearly all phylogenetic papers on freshwater red-algae. As we depend on sequences from GenBank it was mandatory to use them for the sake of comparison with already existing data.
It is necessary to correct the legends of figures and add its forgot about them.
Response: Done.
The most important thing is to support in a more robust way the morphological differences between species and genera.
Response: We have tried to elaborate all available morphological data from the different genera of Batrachospermum s.l. This is, however, difficult as many genera have no clear morphological autapomorphies. Kumanoa is an exception as it has twisted carpogonia. Most of the genera are based on Sirodot’s sections of Batrachospermum and sometimes morphologically weakly defined.
Other observations are highlighted in the manuscript
Response: We have addressed the relevant comments in the manuscript (attached)
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsIn recent decades the freshwater red algal genus Batrachospermum has been split into more than ten genera. In this article the authors describe a new genus and species, Hoefkenia hunsrueckensis, which is morphologically similar to Virescentia vogesiaca but forms a sister clade to the genus Kumanoa. Thus, the erection of the new genus and species is justified.
In general, this article is an important contibution to the taxonomy of Batrachospermum sensu lato and I recommend its publication in the "Diversity".
However, the MS requires significant revision because it has a lot of technical mistakes (see attached file). Also, I think the generic name doesn't quite match the recommendation 20A.1 (h) of the ICN. Since, "Diversity" is not "Phytotaxa" the citations of all works, where the taxa were descibed, seem to be redundant.
Comments for author File: Comments.pdf
Author Response
In recent decades the freshwater red algal genus Batrachospermum has been split into more than ten genera. In this article the authors describe a new genus and species, Hoefkenia hunsrueckensis, which is morphologically similar to Virescentia vogesiaca but forms a sister clade to the genus Kumanoa. Thus, the erection of the new genus and species is justified.
Response: we highly appreciate that you share this view.
In general, this article is an important contribution to the taxonomy of Batrachospermum sensu lato and I recommend its publication in the "Diversity".
However, the MS requires significant revision because it has a lot of technical mistakes (see attached file). Also, I think the generic name doesn't quite match the recommendation 20A.1 (h) of the ICN. Since, "Diversity" is not "Phytotaxa" the citations of all works, where the taxa were described, seem to be redundant.
Response: We have addressed all comments of Reviewer 3 in the attached manuscript. We have reduced the citations of the works where the taxa were described to a necessary minimum. We have checked and corrected the mistakes highlighted by the reviewer in the manuscript. Concerning the name of the new genus, we were aware of Recommendation 20A(h). However, Ulrike Höfken has studied biology and agronomy, and as in her function as Minister of Environment, she has done much more for the conservation of freshwater red-algae than many other authors after whom new genera were named. So we prefer to keep the name Hoefkenia, also to encourage conservation of these highly sensitive organisms.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsBased on the reply to reviewer comments, the authors did not make any change on the original version but merely refused all my concerns and criticisms with weak and unconvincing arguments.
Thus, my conclusions are essentially the same: 1) data set is not convincing at all that the European material represents a new genus; when proposing a new genus, all closely related taxa should be included in the analysis to prove that it does not fit within any previously described taxa and to clearly show the phylogenetic affinities; simply mention that previous studies included all taxa of Kumanoa is fallacious; 2) addition of all European species of Kumanoa, as well as other species of the genus, is mandatory; the current data set only shows that the European material is neighbor of the Kumanoa clade, but does not unequivocally shows that it is a new genus; the reluctancy of the authors to include a large number of species of Kumanoa most probably reflects their fear that their material will be positioned right in the middle of that genus; 3) the proposal of a new genus is a serious matter and should be conducted with the essential requisites, as outlined above.; 4) authors should note that the European Kumanoa virgato-decaisneana (type species of the genus) has its only DNA sequence based on a North American species; I have recently examined specimens of Kumanoa from Germany that closely resembles the one described in this manuscript, and I have reasons to believe the supposedly new genus is merely the "true" K. virgato-decaiseana; authors are highly recommended to prepare a new manuscript including more European material in collaboration with other investigators and do not insist to weak proposal of a new genus.
I strongly recommended rejection of this manuscript, since it has serious flaws and clearly represents bad Science.
Author Response
Based on the reply to reviewer comments, the authors did not make any change on the original version but merely refused all my concerns and criticisms with weak and unconvincing arguments.
Response: The comments of the reviewer became more hostile and indicate a serious conflict of interest.
Thus, my conclusions are essentially the same:
1) data set is not convincing at all that the European material represents a new genus; when proposing a new genus, all closely related taxa should be included in the analysis to prove that it does not fit within any previously described taxa and to clearly show the phylogenetic affinities; simply mention that previous studies included all taxa of Kumanoa is fallacious
Response: Considering the statement that the phylogenetic analyses based on DNA sequences were not properly conducted/presented and not convincing is highly irritating, as state-of-the-art (BI. ML) methods were used for the phylogenetic inferences and clade support estimates. The notion that a phylogram should be presented is puzzling as it is provided in the figure. The rectangular version of the tree was choosen in order to properly visualize the important clade support and the phylogram is depicted exactly to show the length of the branches separating Kumanoa and the new genus Hoefkenia.
2) addition of all European species of Kumanoa, as well as other species of the genus, is mandatory; the current data set only shows that the European material is neighbor of the Kumanoa clade, but does not unequivocally shows that it is a new genus; the reluctancy of the authors to include a large number of species of Kumanoa most probably reflects their fear that their material will be positioned right in the middle of that genus;
Response: This is an insulting remark that disqualifys the reviewer. We have already explained that we have included all clades of Kumanoa in the phylogenetic tree, and especially the European ones. As outlined above a comprehensive phylogeny of Kumanoa has been published by Vis et al. (2012) that we extended almost reaching taxonomic completion of the genus (Fischer et al. 2020). Here we have included representatives of all clades within the genus according to Vis et al. (2012) and Fischer et al. (2020). For Virescentia and Paludicola we have enlarged the sampling adding all available species including our own collections. The outlined species by the reviewer Kumanoa globospora and K. virgatodecaisneana were already included in the analyses as representatives of a derived Kumanoa clade. As to K. abilii, Vis et al. (2012), Necchi et al. (2010) and Fischer at al. (2020) have already shown that this species is part of the K. rwandensis / K. ambigua clade (see below) which we represented by K. rwandensis. Thus, this taxon is deep inside Kumanoa far away from the new taxon. Similarly, K. mahlacensis is deeply nested inside Kumanoa and thus of no concern. As commented above, a generic inclusion of the new taxon in Kumanoa would render other genera like Virescentia, Paludicola and Visia as synonyms of Kumanoa .The K. rwandensis / K. ambigua clade and the position of K. abilii is shown already by Fischer et al. 2020. Plant and Fungal Systematics (65: 147–166, 2020).
3) the proposal of a new genus is a serious matter and should be conducted with the essential requisites, as outlined above.
Response: We agree with this, and we have provided molecular and morphological evidence (e.g. that Hoefkenia hunsrueckensis has no twisted or spirally coined carpogonia and thus lack a morphological autapomorphy of Kumanoa).
All European Kumanoa species are inside the maximally supported monophyletic genus Kumanoa. Hoefkenia hunsreckensis is sister to Kumanoa and genetically and morphologically very distinct from Kumanoa which supports the decision of erecting a genus for the ignored taxon. The main distinguishing character of Kumanoa is the twisted or spirally coiled carpogonial branch while that of Virescentia and the new taxon is straight. Thus, the new taxon does not share the derived characters of Kumanoa. Table 3 gives an overview of the morphological differences between the new Hoefkenia and Kumanoa, Virescentia, Visia and Paludicola. It shows that the morphological differences are often not very marked
4) authors should note that the European Kumanoa virgato-decaisneana (type species of the genus) has its only DNA sequence based on a North American species; I have recently examined specimens of Kumanoa from Germany that closely resembles the one described in this manuscript, and I have reasons to believe the supposedly new genus is merely the "true" K. virgato-decaiseana; authors are highly recommended to prepare a new manuscript including more European material in collaboration with other investigators and do not insist to weak proposal of a new genus.
Response: Indeed, we had no German material of Kumanoa virgatodecaisneana as the species is extremely rare and was not available. The molecular data of the North-American Kumanoa virgatodecaisneana have been used by all previous authors in all revisions and papers of the genus Kumanoa, and the species is deeply nested within this genus. Our plant does not show the morphological autapomorphy of Kumanoa, i.e. the twisted carpogonial branches. For inclusion of Kumanoa species see our response in the first round. For the European species see the Response above. We have no reasons to “believe” but show hard data in our phylogeny.
I strongly recommended rejection of this manuscript, since it has serious flaws and clearly represents bad Science.
Response: This is an insulting remark that disqualifies the reviewer.
Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors
Find some errors in the writing. It is still not clear to me why the legends in figures 2 and 4 do not appear.
My observations are highlighted in yellow, directly in the manuscript
Comments for author File: Comments.pdf
Author Response
Find some errors in the writing. It is still not clear to me why the legends in figures 2 and 4 do not appear.
Response: All errors highlighted by the reviewer have been corrected. The two scientific names in Fig. 1 that showed orthographic errors have been corrected (Kumanoa virgatdecaisna to Kumanoa virgatodecaisneana, Virescentia viride-brasiliense to Virescentia viride-brasiliensis). we have checked that the legends for Fig. 2 and 3 are there.
My observations are highlighted in yellow, directly in the manuscript
