Phylogeography of Ramalina farinacea (Lichenized Fungi, Ascomycota) in the Mediterranean Basin, Europe, and Macaronesia

Round 1

Reviewer 1 Report

1. Although the article's title refers to "the Old World," the content is primarily from Europe, without any examination of Asia or Africa. It is therefore recommended that the article be revised.

2. The Latin names of the species have irregular italics and abbreviations, which point to diligent revision.

3. Your article's research involves complex groups; hence, it ought to address how to define a species. What are their morphologies' differences and similarities? Do morphological species and systematic species contradict one another?

Author Response

1. Although the article's title refers to "the Old World," the content is primarily from Europe, without any examination of Asia or Africa. It is therefore recommended that the article be revised.

Thank you very much for your recommendation.

 Since other reviewers have also suggested this change, the title have been modified to:

Phylogeography of Ramalina farinacea (lichenized fungi, Ascomycota) in the Mediterranean basin, Europe and Macaronesia

2. The Latin names of the species have irregular italics and abbreviations, which point to diligent revision.

Thank you very much. Done.

3. Your article’s research involves complex groups; hence, it ought to address how to define a species. What are their morphologies’ differences and similarities? Do morphological species and systematic species contradict one another?

The genus Ramalina includes some groups of species (e.g., R. decipiens group) where great plasticity and morphological variability is present, and this variability prevents a precise taxonomic identification. As suggested in discussion, the R. farinacea group should also be subjected to an integrative study in the future to resolve its phylogeny and species delimitation. However, the morphology of R. farinacea, in general, is generally well distinguishable from other Ramalina and the morphological characters used to differentiate this species are clear, which is indicated in the Introduction (lines 62-67). In the case of R. alisisoae, the other species considered in the study, the characters defined by [18] in the its description have been used for its identification. The inclusion of the specimen from Mallorca molecularly identified as R. subfarinacea highlights the problems already described by other authors [63] when identifying morphologically very close species of this genus, as is the case of R. farinacea and R. subfarinacea. Therefore, we do not consider it necessary to include the morphological identification of the other species considered in the study, since species delimitation is not the objective of this work and this problematic issue is mentioned in Introduction. In fact, we will deal with these problems in an upcoming work.

Author Response File: Author Response.docx

Reviewer 2 Report

I only have minor comments for this paper and I recommend accept with minor changes, see below:

nrITS should not be italicised

Paragraph 93-103 should be past tense, for example: we specifically tested

Author Response

1. nrITS should not be italicised

Thank you very much. Done.

2. Paragraph 93-103 should be past tense, for example: we specifically tested

Thank you very much. Done.

Author Response File: Author Response.docx

Reviewer 3 Report

This is overall a sound manuscript.

However, there are a few wrong things that really need to be changed, so I go for major revision, even though the revisions I would require would take only a few hours.

First of all, the words "old world" in the title should be replaced by "Europe and Macaronesia", because the study is restricted to this area. The Old World is an area c 50 times larger, including Africa, Asia, Australia. It is ludicrous to claim that you investigate a species complex all over the old world by looking only at two or three percent of area.

Also, the title of a paper does not need a dot at the end. it is not a sentence. so, remove dot after the corrected "Europe and Macaronesia"

Secondly, the claim that Ramalina farinacea is a new report for Cabo Verde is wrong. It has been reported repeatedly from that group of Islands, e g. in:

A. Aptroot & F. Schumm. 2008. Key to Ramalina species known from Atlantic islands, with two new species from the Azores. Sauteria 15: 21-57.

The authors should refer to this publication, which is in any case the most recent monograph of Ramalina in the investigated area, so it is strange that it was not consulted.

in line 62 ff the introduction talks almost only about Swinscow and Krogs assessment about the farinacea group. Missing and needed to be added is a long paragraph over the previously accepted chemically and partly biogeographically different strains within farinacea which even have been accepted for a long time at species level, for instance as hypoprotocetrarica. References are e.g. Culbersons papers in the 70s.

 

small details: italicazation of scientific names is often missing, e.g. in lines 241, 242, 245, 259, 261, 263, 268, 281, 282, 293, 297, 298, 300, 313, 314, 316, 317, 318, 319, 320, 321, 323, 369, 371, 374, 378, 381, 382, 383, 385

line 21: ends with an apostrophe which needs to be deleted; not English

line 333 ends with 2 dots; one is enough

 

Author Response

This is overall a sound manuscript.

However, there are a few wrong things that really need to be changed, so I go for major revision, even though the revisions I would require would take only a few hours.

1. First of all, the words "old world" in the title should be replaced by "Europe and Macaronesia", because the study is restricted to this area. The Old World is an area c 50 times larger, including Africa, Asia, Australia. It is ludicrous to claim that you investigate a species complex all over the old world by looking only at two or three percent of area. Also, the title of a paper does not need a dot at the end. it is not a sentence. so, remove dot after the corrected "Europe and Macaronesia"

Thank you very much for your recommendation.

Since other reviewers have also suggested this change, the title have been modified to:

Phylogeography of Ramalina farinacea (lichenized fungi, Ascomycota) in the Mediterranean basin, Europe and Macaronesia.

2. Secondly, the claim that Ramalina farinacea is a new report for Cabo Verde is wrong. It has been reported repeatedly from that group of Islands, e g. in: Aptroot & F. Schumm. 2008. Key to Ramalina species known from Atlantic islands, with two new species from the Azores. Sauteria 15: 21-57.

The authors should refer to this publication, which is in any case the most recent monograph of Ramalina in the investigated area, so it is strange that it was not consulted.

Although the reference mentioned is included in the manuscript [17], the fact is that we had not noticed the citation of this species in Cape Verde. Thank you very much for your indication, we have modified the text and, now, we do not refer to the Canary Islands as the southern limit of the distribution of R. farinacea.

1. In line 62 ff the introduction talks almost only about Swinscow and Krogs assessment about the farinacea group. Missing and needed to be added is a long paragraph over the previously accepted chemically and partly biogeographically different strains within farinacea which even have been accepted for a long time at species level, for instance as hypoprotocetrarica. References are e.g. Culbersons papers in the 70s.

As the reviewer indicates, and was mentioned in the Introduction, the Ramalina farinacea complex presents a great chemical and ecological variability that has led to several studies aimed at its differentiation. Although the study of Culberson in the 1970s is not cited, we referenced the study of Arroyo and Manrique (1988), where the work of Culberson is cited and the presence and distribution of several chemotypes in the Iberian Peninsula are discussed. Our intention in the future will be to carry out a broader study to delimit the species of the complex and in such study the chemical characterization will be considered due to its taxonomic importance. However, since the objective of the present study is not the delimitation of species of the group, we consider that it would not be necessary to include such information.

2. small details: italicization of scientific names is often missing, e.g. in lines 241, 242, 245, 259, 261, 263, 268, 281, 282, 293, 297, 298, 300, 313, 314, 316, 317, 318, 319, 320, 321, 323, 369, 371, 374, 378, 381, 382, 383, 385.

Done

3. line 21: ends with an apostrophe which needs to be deleted; not English

Done

4. line 333 ends with 2 dots; one is enough

Done

Author Response File: Author Response.docx

Reviewer 4 Report

Dear authors,

Congratulations with a well-crafted study, which I found interesting. It provides some refreshing insights into biogeography of a fungi with a range, indicating close association with evolution of the fascinating Macaronesian flora.

I have just a few suggestions for you to consider. On lines 73-74 in Introduction it was not clear to me, what do you mean as “genetic diversity”. Usually, the main interest is in genetic diversity within populations, especially for purposes of conservation. Your study has several main objectives: (i) interspecific phylogenetic relationships; (ii) intraspecific phylogeography of R. farinacea; (iii) genetic structure within the species; (iv) comparison of genetic diversity of sampled groups of specimens of the species from different regions across Europe. The whole study is structured along these objectives. Please state this explicitly in the Introduction.

 

Further, given your results, it is equally probable that R. farinacea evolved in western Mediterranean and later diversified into two clades (probably through vicariance), one of which had been mostly restricted to Macaronesia. With the data at hand, we cannot give preference to any of these hypotheses with confidence. This uncertainty should be reflected in your final statements.

I also think that you should elaborate a bit on the indications of multiple cases of colonization of Macaronesia by R. farinacea. These indications can be seen in your results, so I wondered why were you not discussing them?

Below you can find my minor comments.

L. 108 What is “SPO-IPV”? Please spell out or explain.

L. 123-125. Please justify using these specific loci. Why exactly these two loci? Why not one or 100 loci? Usage of ITS is relatively well-known in phylogenetic analyses. This cannot be told about the second gene. What is so special about it that you decided to base your analyses on its sequences?

L. 152. Why were outgroups not used in alignments? What was the reason for including outgroups in your analyses, if they were not aligned with the ingroup?

L. 163-164. Usually, significantly supported are considered nodes with support values above 95% probability. Support at the level of 70% is weak.

L. 164. Please specify the version of MrBayes software which you used in your analyses.

L. 197-202. The title of this sub-chapter announced analyses of neutrality. Please either describe these analyses, or exclude “neutrality” from the title.

L. 208-209. Are the two genera with known substitution rates phylogenetically close to Ramalina? Please clarify. If they are not, using substitution rates from these genera may be misleading.

l. 222-223. Why PCR filed in exactly the same specimens? Please clarify meaning of this sentence.

L. 230. What does this mean: “…branching pattern was no overally bifurcating”? Would not it be clearer to say simply that proportion of polytomies and weakly supported nodes in the tree was high.

L. 232. Please provide information on geographic origin of the listed here specimens.

L. 313. I suggest a slight modification of this sentence: “The origin and diversification…started in the late Miocene…” Your results do not support the idea of the middle Miocene origin of R. farinacea.

L. 379. “The distinct signals…were also revealed”, plural.

L. 385. Grammar: “did not form… in either of the three….”

L. 387. Grammar again: “Phylogenetic conflicts…”, plural. Please note that if you are using the plural verbs “are” or “were”, these verbs should correspond (here and likely in several other occasions} to plural nouns.

L. 411. What “were be” means? Please use grammatically correct and clear phrases. In this case, “could be” would do.

L. 427. Grammar, please correct: “agree with…”, or “are in agreement with…”.

L. 432-434. Please note that dating analyses cannot be used to support hypotheses on areas of origin of taxa or clades. This should be done by estimating ancestral area probabilities for specific clades.

L. 464-466. Given your results, it is equally probable that R. farinacea evolved in western Mediterranean and later diversified into two clades (probably through vicariance), one of which had been mostly restricted to Macaronesia. With the data at hand, we cannot give preference to any of these hypotheses with confidence.

Best regards,

Author Response

Dear authors,

Congratulations with a well-crafted study, which I found interesting. It provides some refreshing insights into biogeography of a fungus with a range, indicating close association with evolution of the fascinating Macaronesian flora.

I have just a few suggestions for you to consider. On lines 73-74 in Introduction it was not clear to me, what do you mean as “genetic diversity”. Usually, the main interest is in genetic diversity within populations, especially for purposes of conservation.

Genetic diversity of a given species can be evaluated for multiple purposes, not only conservation. We in fact used two genetic markers to explore how the diversity of R. farinacea at the genetic level is structured geographically. By such exploration we have been able to test a particular hypothesis about the geographic origin of the species. Other works that have focused on the same objective and therefore used the same or similar methodological and conceptual approaches were, for example:

Werth, S., & Sork, V. L. (2010). Identity and genetic structure of the photobiont of the epiphytic lichen Ramalina menziesii on three oak species in southern California. American Journal of Botany, 97(5), 821-830.

Fernández‐Mendoza, F., Domaschke, S., García, M. A., Jordan, P., Martín, M. P., & Printzen, C. (2011). Population structure of mycobionts and photobionts of the widespread lichen Cetraria aculeata. Molecular Ecology, 20(6), 1208-1232.

Widmer, I., Dal Grande, F., Excoffier, L., Holderegger, R., Keller, C., Mikryukov, V. S., & Scheidegger, C. (2012). European phylogeography of the epiphytic lichen fungus L obaria pulmonaria and its green algal symbiont. Molecular Ecology, 21(23), 5827-5844.

Fernández‐Mendoza, F., & Printzen, C. (2013). Pleistocene expansion of the bipolar lichen C etraria aculeata into the S outhern hemisphere. Molecular Ecology, 22(7), 1961-1983.

Sork, V. L., & Werth, S. (2014). Phylogeography of R amalina menziesii, a widely distributed lichen‐forming fungus in western N orth A merica. Molecular Ecology, 23(9), 2326-2339.

Onuţ‐Brännström, I., Tibell, L., & Johannesson, H. (2017). A worldwide phylogeography of the whiteworm lichens Thamnolia reveals three lineages with distinct habitats and evolutionary histories. Ecology and Evolution, 7(10), 3602-3615.

These works (and many others) are seminal in lichen phylogeography and have shed much light into the geographic origin of species and the timing of their diversification and geographical range changes.

Your study has several main objectives: (i) interspecific phylogenetic relationships; (ii) intraspecific phylogeography of R. farinacea; (iii) genetic structure within the species; (iv) comparison of genetic diversity of sampled groups of specimens of the species from different regions across Europe. The whole study is structured along these objectives. Please state this explicitly in the Introduction.

Thank you very much for the suggestion. We have modified a paragraph of the Introduction (see new lines 102-107) to clarify the objectives as indicated by the referee. 

Further, given your results, it is equally probable that R. farinacea evolved in western Mediterranean and later diversified into two clades (probably through vicariance), one of which had been mostly restricted to Macaronesia. With the data at hand, we cannot give preference to any of these hypotheses with confidence. This uncertainty should be reflected in your final statements.

Ok. We have modified the text either in Abstract and Discussion section in a more cautious way. Now, the concerns raised by the Reviewer are explicitly written.

I also think that you should elaborate a bit on the indications of multiple cases of colonization of Macaronesia by R. farinacea. These indications can be seen in your results, so I wondered why were you not discussing them?

We refrained from discussing this topic because our results cannot distinguish between the two possibilities of dispersal taking place from (1) the Mediterranean towards Macaronesia, or (2) vice versa. To do this we must need even a more thorough specimen and molecular dataset and test plausible hypotheses of dispersal in programs as for example Migrate, which calculates migration under a Bayesian (or ML) frameworks (e.g., Garrido-Benavent et al. 2018-Journal of Biogeography).

Below you can find my minor comments.

1. 108 What is “SPO-IPV”? Please spell out or explain.

SPO-IPV referred to a citation. It has been replaced by the number.

125. 123-125. Please justify using these specific loci. Why exactly these two loci? Why not one or 100 loci? Usage of ITS is relatively well-known in phylogenetic analyses. This cannot be told about the second gene. What is so special about it that you decided to base your analyses on its sequences?

As the referee indicates, the use of the ITS (considered as a barcode in fungi as specified in the text line 130) is widely accepted. To be able to compare with any database it is necessary to include the ITS in phylogenetic studies.

Regarding the uid70 marker, in chapter 1 (“Endless forms most frustrating: disentangling species boundaries in the Ramalina decipiens group (Lecanoromycetes, Ascomycota”), with the description of six new species and a key to the group) of the recently defended doctoral thesis of Miguel Blázquez (co-author of this study): “Evolution of the Ramalina decipiens group (lichenized Ascomycota) in Macaronesia: comparative study of its symbionts and ecophysiological traits”, it is explained in detail how this marker has been selected and what characteristics make it useful for the description of species of Ramalina. Although the work has not yet been published, the thesis has already been defended and is cited when reference is made to the marker. However, following the Referee suggestions we have now included a new sentence to justify its use in Material and methods (see line 131).

152. 152. Why were outgroups not used in alignments? What was the reason for including outgroups in your analyses, if they were not aligned with the ingroup?

We conducted two sets of phylogenetic analyses. In section 2.3 of Material and methods we did not used sequences for rooting purposes because: (1) the phylogenies were inferred in order to explore specimen clustering and compare if resulting groupings were consistent among phylogenies (we have now explicitly mentioned this at the beginning of this section (see lines 155-157)); and (2) because we still lack uid70 data for taxa that could be regarded as suitable outgroups, and therefore we did not root phylogenies for coherence, avoiding that the nrITS phylogeny was rooted and the other not.

Then, in section 2.6 (Dating Analyses) we did use an outgroup for rooting purposes because we needed to determine well the evolutionary relationships of the ingroup, as it would allow use to obtain crucial insights into the temporal and spatial diversification of R. farinacea. Furthermore, the use of outgroup sequences in dating analyses is customary in lichen phylogeographical studies (e.g., Fernández-Mendoza & Printzen 2013-Molecular Ecology; Garrido-Benavent et al. 2018- Journal of Biogeography, 2021-Molecular Phylogenetics and Evolution).

164. 163-164. Usually, significantly supported are considered nodes with support values above 95% probability. Support at the level of 70% is weak.

Well, it depends on whether the support is obtained in a Maximum Likelihood or a Bayesian framework. There is a “general agreement” in considering values of nodal bootstrap support of 70% or greater as “significant support”. Of course, BS values of 95% are much better than 70% but, still, many research articles (if not all) in the (fungal) literature that inferred phylogenies consider 70% as the “starting point” for supported nodes. We followed this “rule”, although we agree with the Referee that 70% is weak support (but, still, it should be considered “enough” support).

In the case of Bayesian phylogenies, like those inferred with MrBayes, we consider as supported nodes only those that have posterior probabilities (PP) equal or greater than 0.95 (being 1 the highest possible value).

164. 164. Please specify the version of MrBayes software which you used in your analyses.

Ok, done. It was v. 3.2.6.

202. 197-202. The title of this sub-chapter announced analyses of neutrality. Please either describe these analyses, or exclude “neutrality” from the title.

Ok, done.

209. 208-209. Are the two genera with known substitution rates phylogenetically close to Ramalina? Please clarify. If they are not, using substitution rates from these genera may be misleading.

No, they belong into different families (Ramalinaceae vs Parmeliaceae). There are two reasons why we used these substitution rates:

• In general, lichens lack a suitable and varied fossil record, and therefore it is usually impossible to use find out particular fossils that allow for primary calibrations in lichen dating studies. The lack of a varied fossil record is extensible to most fungi, not only the lichenized ones.
• There aren’t specific substitution rates calculated for any member of Ramalinaceae in the lichen literature. Because of this lack, we had to use substitution rates of two genera that belong into family Parmeliaceae. However, it is important to note that Ramalinaceae and Parmeliaceae share the same taxonomic order (Lecanorales). In lichen phylogeographical studies, researches often use available substitution rates for dating irrespective of the closeness of the focal species and the ones with the available rates (e.g., Onuţ‐Brännström et al. 2017-Ecology and Evolution; Garrido-Benavent et al. 2018-Journal of Biogeography; Chiva et al. 2019-Journal of Biogeography).

We are aware of the potential bias of using substitution rates from unrelated lineages to estimate divergence times in the R. farinacea. However, our study provides a valuable hypothesis of the timing of diversification that merits additional study. We have added a similar sentence in Discussion (see lines 470-472).

223. 222-223. Why PCR filed in exactly the same specimens? Please clarify meaning of this sentence.

They are not the same specimens, perhaps due to a possible misleading in the sentence the referee has understood that they are the same and therefore we have decided to remove it. In addition, in material and methods (line 161) it is indicated that the MSA of the ITS contains 106 and that of the uid70 contains 88.

230. 230. What does this mean: “…branching pattern was no overally bifurcating”? Would not it be clearer to say simply that proportion of polytomies and weakly supported nodes in the tree was high.

Done.

232. 232. Please provide information on geographic origin of the listed here specimens.

Done.

313. 313. I suggest a slight modification of this sentence: “The origin and diversification…started in the late Miocene…” Your results do not support the idea of the middle Miocene origin of R. farinacea.

Done.

379. 379. “The distinct signals…were also revealed”, plural.

Done

385. 385. Grammar: “did not form… in either of the three….”

Done.

387. 387. Grammar again: “Phylogenetic conflicts…”, plural. Please note that if you are using the plural verbs “are” or “were”, these verbs should correspond (here and likely in several other occasions} to plural nouns.

Done.

411. 411. What “were be” means? Please use grammatically correct and clear phrases. In this case, “could be” would do.

Thank you very much. The sentence has been deleted.

427. 427. Grammar, please correct: “agree with…”, or “are in agreement with…”.

Done.

434. 432-434. Please note that dating analyses cannot be used to support hypotheses on areas of origin of taxa or clades. This should be done by estimating ancestral area probabilities for specific clades.

We have modified the Discussion paragraph and now we do not explicitly support a given hypothesis, as recommended by the Referee. We are aware of the usefulness of reconstructing ancestral areas (we do know several approaches to do it) but we did not conduct it because the support of the inner clades in our phylogeny is not great and therefore any analysis of reconstructing ancestral areas may be misleading. We will prepare a more robust phylogeny in future adding more markers and will then apply this type of approaches to delve further on the geographic origin of R. farinacea and close allies.

466. 464-466. Given your results, it is equally probable that R. farinacea evolved in western Mediterranean and later diversified into two clades (probably through vicariance), one of which had been mostly restricted to Macaronesia. With the data at hand, we cannot give preference to any of these hypotheses with confidence.

Answered previously.11

 

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

This has been satisfactorily improved

 

the abstract contains words with hyphens in the middle. remove them