Biogeography and Diversification of Bumblebees (Hymenoptera: Apidae), with Emphasis on Neotropical Species

Round 1

Reviewer 1 Report

The manuscript “Biogeography and diversification of bumblebees (Hymenoptera: Apidae), with emphasis on Neotropical species” reports an updated phylogeny with divergence times for the genus Bombus. The paper adds a few new species and uses new fossils to calibrate the phylogeny, but otherwise largely recapitulates previously found phylogeographic patterns in the genus. The increased focus on South American species could be useful for other researchers working on these species.  I think the study is publishable but there needs to be considerable changes to the methods and presentation of results before I would consider acceptance.

Major comments:

1. I am not able to open many of the supplementary figures as the files are damaged, so I am unable to completely assess the manuscript. Many of the supplemental figures have huge file sizes, so the authors might consider reformatting those if possible.

2. I have a few concerns about some of the bioinformatics here. Specifically, why were sequences concatenated prior to bioinformatic alignment? Is this necessary because of the relatively small amount of data? I think, at least, that mitochondrial and nuclear phylogenies should be estimated separately given that substitution patterns necessarily differ between the two genomes. I know that this is done in many studies, but that doesn’t mean it’s a good idea if it can be avoided. Another possibility to improve the analysis would be to select fourfold degenerate sites, which are most likely to be evolving neutrally – although perhaps that would also leave the authors with too little data? I would love to see some reanalysis here, and if not, I would expect to see substantial justification of the current methodologies in the revised ms.

3. Table 2 has a lot of problems. First of all, each line of the table should refer to a divergence event, not a species. There is not a separate speciation event, for example, for morio and B. dahlbomii, so this should be represented by one line in the table. Second, the caption specifies that the abbreviation OBS means observation, but I don’t see any OBS in the table, so I wonder if it’s missing something. Third, if the authors wish to hypothesize a geologic cause of vicariance events, they should be free to do so, but it should be clear that this is speculation. The title of the column “Cause event” makes it seem like the authors have substantive support for these causes. Third, many of the causes don’t even make sense as hypotheses! To pick one example, “Andes uplift” is given as a cause for the split between B. pullatus and B. pauloensis. But their divergence occurred a maximum of 6.2 Ma, whereas my understanding is that the Andean orogeny was nearly if not totally complete by this time. Even if the timing made sense, Figures 1 and 2 suggest that these species both exist on both sides of the Andes (regions A and B)! Further given causes, such as “climate oscillations” and “marine transgression” are vague, and it’s not at all clear to me why they would cause speciation among bees. The formation of the Isthmus of Panama certainly shouldn’t cause vicariance of terrestrial species. I would suggest that the authors remove this column from the table. Predicting vicariance events is hard, and it’s clear the authors don’t have strong hypotheses for most of these speciation events, and that’s okay! They should just admit that and possibly highlight a few in the text that maybe do correspond more logically with the phylogeographic data.

4. This discussion is pretty long and tedious, discussing in detail specific hypotheses for migrations of nearly every neotropical lineage. This comes after a lot of very similar detail is also presented at length in the results. I’d suggest that the authors focus on a few of the major results and trends found with the data, as the current version doesn’t really give me a great sense of why the paper is important. A more detailed version of Figure 3 (see also minor comment on this figure below), or even a separate similar figure focused on South America, could show readers some of these detailed patterns without the authors needing to discuss them quite so much.

Minor comments:

Lines 93, 135, 341: Given that this is a molecular phylogeny I would change the word “character/characters” to “locus/loci”

Lines 96-98: What were the new species sequenced? It doesn’t say in Table S1, maybe it does in one of the other supplementals but this really needs to go in the main text. The outcome of the new species and their place in the phylogeny should be highlighted in the results as this is the only new data offered by the paper. I do appreciate that the authors intend to deposit this data to NCBI and indicate so in the data availability statement.

Lines 125-126; 188-191: What are “rogue taxa” and why do they need to be identified if the authors aren’t going to remove them? The authors should either define and explain this in more detail or just leave it out of the manuscript entirely.

Lines 137-139: If these methods are not accurate (as the authors argue), why report them? I would just get rid of the paragraph and appendix, it’s distracting and contributes to the paper being far too long.

Lines 166-167: The authors should provide justification if they are going to depart from using previously defined bioregions in their analysis. 

Lines 297-398: I’m not disputing the point, but why exactly do the current results support this pattern? I’m guessing it has to do with the timing of arrival not coinciding with possible land links via western Europe? It would be worthwhile to briefly justify this to readers, it isn’t obvious.

Figure 1: Can the authors remake the left part of figure 1? The proportions seem wrong and the enormous block sizes are jarring and kind of distracting. There are several good R packages for drawing and shading maps.

Figure 2: Why is this figure split into three parts if the authors are just going to put them right on top of each other? And why isn’t the bottom part of the phylogeny shown? The figure caption says that letters and numbers correspond to figure one, but I don’t see any numbers in either figure.

Figure 3: What do the numbers to the right of the date ranges refer to? I can’t access Figures S2-S5 so maybe that would help, but the figure caption should explain this regardless.

Table 1: Footnote 3 just says exactly what is already in the table. If kept or modified, it should be changed to footnote 2 because it comes before the current footnote 2. Also, in that row “Nearctic” is misspelled.

Appendix C: Given that there is already description of the B. applanatus – B. brevivillus question in the text (Lines 212-217), I’m not sure this merits an appendix. Maybe the authors could briefly add in the text that morphological comparison also supports synonymy of the two species and cite the relevant supplemental figure.

Appendix D: If these results are important enough to be cited in the discussion then I feel they should be included in the Methods and Results sections alongside the rest of the analysis.  I am okay with the missing data analysis (Appendix E) remaining an appendix because it does not produce any core biological results, but is rather a confirmation of methodology.

Author Response

Major comments:

1. I am not able to open many of the supplementary figures as the files are damaged, so I am unable to completely assess the manuscript. Many of the supplemental figures have huge file sizes, so the authors might consider reformatting those if possible.

Yes, there were four figures that were corrupted, thus I replaced them: Figure S2, Figure A2 and Figure A4 were replaced by new PDF files, and D2 by another file. I also added figures in .pdf format for the bigger figures that were not corrupted.

1. I have a few concerns about some of the bioinformatics here. Specifically, why were sequences concatenated prior to bioinformatic alignment? Is this necessary because of the relatively small amount of data? I think, at least, that mitochondrial and nuclear phylogenies should be estimated separately given that substitution patterns necessarily differ between the two genomes. I know that this is done in many studies, but that doesn’t mean it’s a good idea if it can be avoided. Another possibility to improve the analysis would be to select fourfold degenerate sites, which are most likely to be evolving neutrally – although perhaps that would also leave the authors with too little data? I would love to see some reanalysis here, and if not, I would expect to see substantial justification of the current methodologies in the revised ms.

Actually, the sequences were not concatenated prior to alignment, they were aligned and only concatenated later. Different nuclear gene segments, such as exons and introns, were also aligned separately. Sequences from coding regions (COX1 and CytB genes along with the exons of the nuclear genes) were further partitioned into three, 1st, 2nd and 3rd codon bases.

Considering that substitution patterns may differ within and between genomes, we employed two approaches to address this issue: partitioning (in order to account for differences in substitution patterns between gene segments) and a gamma-distributed rate model (in order to account for differences among sites within gene segments). Additionally, the combination of fast and slowly evolving sites allowed us to attain phylogenetic resolution at different depths of the Bombus phylogenetic tree.

1. Table 2 has a lot of problems. First of all, each line of the table should refer to a divergence event, not a species. There is not a separate speciation event, for example, for morio and B. dahlbomii, so this should be represented by one line in the table. Second, the caption specifies that the abbreviation OBS means observation, but I don’t see any OBS in the table, so I wonder if it’s missing something. Third, if the authors wish to hypothesize a geologic cause of vicariance events, they should be free to do so, but it should be clear that this is speculation. The title of the column “Cause event” makes it seem like the authors have substantive support for these causes. Third, many of the causes don’t even make sense as hypotheses! To pick one example, “Andes uplift” is given as a cause for the split between B. pullatus and B. pauloensis. But their divergence occurred a maximum of 6.2 Ma, whereas my understanding is that the Andean orogeny was nearly if not totally complete by this time. Even if the timing made sense, Figures 1 and 2 suggest that these species both exist on both sides of the Andes (regions A and B)! Further given causes, such as “climate oscillations” and “marine transgression” are vague, and it’s not at all clear to me why they would cause speciation among bees. The formation of the Isthmus of Panama certainly shouldn’t cause vicariance of terrestrial species. I would suggest that the authors remove this column from the table. Predicting vicariance events is hard, and it’s clear the authors don’t have strong hypotheses for most of these speciation events, and that’s okay! They should just admit that and possibly highlight a few in the text that maybe do correspond more logically with the phylogeographic data.

We made the adjustments in table 2 and changed the "Cause event" column header to "Suggested events".

Regarding Bombus pauloensis: We put the footnote in the worksheet to make it easier to understand, it was previously just in the text.

"Here it was considered that the B. pauloensis lineage that ranges across the Andes in northern Colombia does not belong to the same species as those from tropical lowlands of Brazil and surrounding countries."

1. This discussion is pretty long and tedious, discussing in detail specific hypotheses for migrations of nearly every neotropical lineage. This comes after a lot of very similar detail is also presented at length in the results. I’d suggest that the authors focus on a few of the major results and trends found with the data, as the current version doesn’t really give me a great sense of why the paper is important. A more detailed version of Figure 3 (see also minor comment on this figure below), or even a separate similar figure focused on South America, could show readers some of these detailed patterns without the authors needing to discuss them quite so much.

Minor comments:

Lines 93, 135, 341: Given that this is a molecular phylogeny I would change the word “character/characters” to “locus/loci”

Done.

Lines 96-98: What were the new species sequenced? It doesn’t say in Table S1, maybe it does in one of the other supplementals but this really needs to go in the main text. The outcome of the new species and their place in the phylogeny should be highlighted in the results as this is the only new data offered by the paper. I do appreciate that the authors intend to deposit this data to NCBI and indicate so in the data availability statement.

It is already listed as "in this article" in table S1. NCBI vouchers will be in the final version of the article.

Lines 125-126; 188-191: What are “rogue taxa” and why do they need to be identified if the authors aren’t going to remove them? The authors should either define and explain this in more detail or just leave it out of the manuscript entirely.

We believe that we should be clear about the issues of phylogenetic analyses, so we decided to keep it.

Lines 137-139: If these methods are not accurate (as the authors argue), why report them? I would just get rid of the paragraph and appendix, it’s distracting and contributes to the paper being far too long.

We decided to keep, as it is important notice for readers.

Lines 166-167: The authors should provide justification if they are going to depart from using previously defined bioregions in their analysis.

The number of areas was decreased (after ignoring the subdivisions of the Nearctic region) in order to circumvent the BioGeoBears total number of areas limitation, while preserving a level of granularity within the Neotropical region that would allow us to discuss the biogeographical patterns in this region.

Lines 297-398: I’m not disputing the point, but why exactly do the current results support this pattern? I’m guessing it has to do with the timing of arrival not coinciding with possible land links via western Europe? It would be worthwhile to briefly justify this to readers, it isn’t obvious.

This conclusion is based on the lack of coincidence between the time of arrival of the species in America and the age of possible connections of land linking Europe and America.

Figure 1: Can the authors remake the left part of figure 1? The proportions seem wrong and the enormous block sizes are jarring and kind of distracting. There are several good R packages for drawing and shading maps.

Done.

Figure 2: Why is this figure split into three parts if the authors are just going to put them right on top of each other? And why isn’t the bottom part of the phylogeny shown? The figure caption says that letters and numbers correspond to figure one, but I don’t see any numbers in either figure.

The figure 2 has been split for ease of viewing, it was very large and difficult to read. A mistake occurred while formatting the journal template and only three parts were inserted. We removed "and numbers" from the subtitle and fixed the mistake.

Figure 3: What do the numbers to the right of the date ranges refer to? I can’t access Figures S2-S5 so maybe that would help, but the figure caption should explain this regardless.

The numbers to the right of the dates are the events indicated in the phylogenies. We explained this in the figure caption.

Table 1: Footnote 3 just says exactly what is already in the table. If kept or modified, it should be changed to footnote 2 because it comes before the current footnote 2. Also, in that row “Nearctic” is misspelled.

Done.

Appendix C: Given that there is already description of the B. applanatus – B. brevivillus question in the text (Lines 212-217), I’m not sure this merits an appendix. Maybe the authors could briefly add in the text that morphological comparison also supports synonymy of the two species and cite the relevant supplemental figure.

Because it is a point not mentioned in other manuscripts, we decided to keep it as is.

Appendix D: If these results are important enough to be cited in the discussion then I feel they should be included in the Methods and Results sections alongside the rest of the analysis.  I am okay with the missing data analysis (Appendix E) remaining an appendix because it does not produce any core biological results, but is rather a confirmation of methodology.

We decided to keep as an appendix as these analyses are not a consensus among researchers, as it is very difficult to infer extinction rate over time.

Reviewer 2 Report

In the article “biogeography and diversification of bumblebees (Hymenoptera: Apidae), with emphasis on Neotropical species”, the authors work to discern the evolutionary relationships, as well as diversification patterns of neotropical bumblebees.  Overall, I really enjoyed reading this manuscript. While there is a fair amount of data on bumblebees available, there is a lot of good information here that may not be available in other resources. There are a few suggestions I have for the manuscript, some of them are more important than others.  I first mention the important suggestions, then follow it with minor suggestions.

Major Suggestions:

Line 98:  I would specify that collection locality and voucher information can be found in this table here rather than just having it in the table without mentioning it. 

Lines ~105-122:  I would suggest breaking this up into multiple sections.  Ex: DNA Extraction, DNA PCR & sequencing DNA alignment and Partition finder

Line 125:  Alignment could possibly be placed in the "phylogenetic inference location".  This is where I would look at least

Line 128:  What models were used?

Lines 184-191:  I would combine these paragraphs.  They are too short to stand on their own in my opinion

Figure 2:  I think this figure needs to be displayed in a different fashion.  It's hard to read with the font size so small.  I would suggest maybe. making these bigger or separating aspects of this figure into multiple figures.  Maybe just a tree with divergence times, and then a paneled figure with the color (needs a legend)

Minor Suggestions:

Throughout the manuscript the authors talk about time ranges (ex:  47- 34 million years ago). I feel the order of the dates is a bit odd.  I feel like they should be flipped (ex: 34-47 million years ago). This feels like a more natural way to present this (smaller to bigger number, especially relative to the present when we talk about “millions of years ago”).  They way it is presented is chronologically correct, but is not termed in dates (ex: 2000-2020).

Line 17:  Maybe just put "...diversification in the Neotropical Region" and omit “particularly”

Line 90:  should be “evolve” and not “evolved”

Author Response

Major Suggestions:

Line 98:  I would specify that collection locality and voucher information can be found in this table here rather than just having it in the table without mentioning it. 

Done.

Lines ~105-122:  I would suggest breaking this up into multiple sections.  Ex: DNA Extraction, DNA PCR & sequencing DNA alignment and Partition finder

We decided to keep it that way, because the paragraphs would be too small.

Line 125:  Alignment could possibly be placed in the "phylogenetic inference location".  This is where I would look at least

Done.

Line 128:  What models were used?

The models are in the results table S6.

Lines 184-191:  I would combine these paragraphs. They are too short to stand on their own in my opinion

Done.

Figure 2:  I think this figure needs to be displayed in a different fashion.  It's hard to read with the font size so small.  I would suggest maybe. making these bigger or separating aspects of this figure into multiple figures.  Maybe just a tree with divergence times, and then a paneled figure with the color (needs a legend)

Done.

Minor Suggestions:

Throughout the manuscript the authors talk about time ranges (ex:  47- 34 million years ago). I feel the order of the dates is a bit odd.  I feel like they should be flipped (ex: 34-47 million years ago). This feels like a more natural way to present this (smaller to bigger number, especially relative to the present when we talk about “millions of years ago”).  They way it is presented is chronologically correct, but is not termed in dates (ex: 2000-2020).

I'm sorry, we disagree and prefer to keep it as is.

Line 17:  Maybe just put "...diversification in the Neotropical Region" and omit “particularly”

Done.

Line 90:  should be “evolve” and not “evolved”

Done.

Reviewer 3 Report

The manuscript on the biogeography and diversification of bumblebees presents a very interesting and fascinating approach towards understanding how bumblebees speciated and expanded over time. The authors employ a great variety of modern approaches to come to their conclusions, and in general the manuscript is very well written. There are however a few points that the authors need to address before it can be accepted for publication (see below). The most important among them, is some clarifications that would definitely increase the insight and clarity of their work.

Line 340: ...within them...

Lines 371-372: Did you want to say here "....that this was not improbable."? Please check carefully the content.

Line 415: What do you mean here by "nuclear"? I guess that here went something wrong - please check it again.

Line 448-453: Here I suggest that you enahnce mor the arguments on why there is a discrepancy between the dates that you have come to and the dates suggested by Hines in the previous study.

Line 462: ...existing data indicate...

Line 465:...stepping stones...

Figure 3: I believe that this figure should be thoroughly reconstructed, as it includes the main outcome of this very exciting work. The authors should try to make it more self-explanatory, as in its current form, it is impossible for the reader to follow the background information.

Author Response

Line 340: ...within them...

Done.

Lines 371-372: Did you want to say here "....that this was not improbable."? Please check carefully the content.

No. The extinction of taxa of some groups in a few regions can make it difficult to interpret how a lineage arrived in an area without crossing through the intermediate areas.

Line 415: What do you mean here by "nuclear"? I guess that here went something wrong - please check it again.

Done.

Line 448-453: Here I suggest that you enahnce mor the arguments on why there is a discrepancy between the dates that you have come to and the dates suggested by Hines in the previous study.

The discrepancy among inferred dates here and the Hines' suggested can be explained through the different methods and priors were used to assess the time of diversification of extant Bombus lineages. While Hines used the rates of evolution from the literature or just a few calibration points we used nine calibration points going from Late Cretaceous (Cretotrigona prisca (Michener & Grimaldi, 1988) - 70.6–66.043 Ma) to Middle Miocene (Apis lithohermaea Engel, 2006 - 15.97–13.65 Ma). The discussion is already too long according to one of the reviewers, so we decided to keep it as it is.

Line 462: ...existing data indicate...

Done.

Line 465:...stepping stones...

Done.

Figure 3: I believe that this figure should be thoroughly reconstructed, as it includes the main outcome of this very exciting work. The authors should try to make it more self-explanatory, as in its current form, it is impossible for the reader to follow the background information.

We'll leave it as is, but we've improved the subtitles.

Round 2

Reviewer 1 Report

I am reviewing the manuscript “Biogeography and diversification of bumblebees (Hymenoptera: Apidae), with emphasis on Neotropical species”, for the second time. I initially thought that the manuscript needed significant changes before I could recommend publication. The authors have made only very superficial changes in response to some of my comments, and have chosen to simply not address others. At many points, the authors responded to my comments but did not incorporate those responses into the ms itself, which they need to do. I therefore feel that the manuscript is not much closer to publication and I would still like to see the authors address several points by making changes to the text and figures.

Major comments:

1. I was concerned in the first draft about the concatenation of nuclear and mitochondrial data prior to phylogenetic and divergence time analysis, given that these have necessarily different mutation rates and demographic histories. I suggested that the authors could leave the analysis as is if they could provide a convincing justification for the validity and accuracy of the method given their data. Although the authors have provided an explanation in their response to my review, they have not changed anything in the manuscript itself. These justifications need to be in the paper or else they serve no purpose. There are details in that response that are not even hinted at in the manuscript, which further highlights that the work is not reproducible as written.

2. Table 2 has improved slightly. I still don’t see any justification in the text for the hypothesis that the raising of a land bridge could cause a vicariance event between two terrestrial insect species. It just doesn’t make any sense. “Climatic oscillations” still seems like such a vague handwave that I would prefer to leave it out, it’s really not a meaningful hypothesis even as speculation.

3. I’m a bit confused by the authors’ refusal to include the species they have provided new sequence for in the main text. I do see where they have indicated the newly sequenced species in Table S1, but that’s pretty difficult to find unless you read that long table closely. They should be proud of generating new data, and the description of that new data needs to be in the main text. There also needs to be at least a cursory description of the findings of the new data collection in the results section. None of this would add more than a few very simple sentences.

Other comments:

1. Appendix C is pointless and should be removed. The purpose of the paper is to make information easily accessible to readers. Adding unnecessary appendices and supplementary materials places an undue burden on readers.

2. I commented on the use of the term “rogue taxa” and whether it was necessary to include its description if they chose not to remove them. I don’t know what rogue taxa are and many readers won’t either. The authors need to explain this further.

Lines 295-296, 534-536: The authors claim that B. pauloensis should be considered as two different species on either side of the Andes, which neatly fits their hypothesis that “Andes uplift” is responsible for the vicariance event between B. pauloensis and B. pullatus. However, they provide no justification for this other than a vague and uncited claim that “several taxonomists suspect” that they are different species. This is a scientific paper and claims need to be supported by evidence. If the authors can provide evidence for this then the hypothesis still makes no sense – clearly, “Andes uplift” did not separate these two species but rather the two cryptic B. pauloensis species.

Table 1: remove space between B. and Brachycephalus in line 5.

Figure 2: Please remove the legend, as no reader could possibly use this legend to distinguish between these colors and glean any information about the figure.

Figure 2/3: I appreciate that the numbers in Figure 3 have been explained but I don’t see any events indicated by corresponding numbers in Figure 2, so I still don’t see what information they give us.

Author Response

4. This discussion is pretty long and tedious, discussing in detail specific hypotheses for migrations of nearly every neotropical lineage. This comes after a lot of very similar detail is also presented at length in the results. I’d suggest that the authors focus on a few of the major results and trends found with the data, as the current version doesn’t really give me a great sense of why the paper is important. A more detailed version of Figure 3 (see also minor comment on this figure below), or even a separate similar figure focused on South America, could show readers some of these detailed patterns without the authors needing to discuss them quite so much.

We reduced some of the comments in Discussion, as indicated.

Major comments:

1. I was concerned in the first draft about the concatenation of nuclear and mitochondrial data prior to phylogenetic and divergence time analysis, given that these have necessarily different mutation rates and demographic histories. I suggested that the authors could leave the analysis as is if they could provide a convincing justification for the validity and accuracy of the method given their data. Although the authors have provided an explanation in their response to my review, they have not changed anything in the manuscript itself. These justifications need to be in the paper or else they serve no purpose. There are details in that response that are not even hinted at in the manuscript, which further highlights that the work is not reproducible as written.

What you requested is implicit in our decision to partition the data, as mentioned in the first paragraph of the PartitionFinder article:

"In phylogenetic analyses it is important to account for variation in rates and patterns of evolution among sites (Yang 1996; Kumar et al. 2012). Partitioning attempts to achieve this by estimating independent models of molecular evolution for subsets of sites that are deemed to have evolved in similar ways. It can be challenging to choose a good partitioning scheme, because the number of possible schemes can be extremely large."

All data are provided in the supplementary material to allow reproducing the results presented here (e.g. inputs and output).

1. Table 2 has improved slightly. I still don’t see any justification in the text for the hypothesis that the raising of a land bridge could cause a vicariance event between two terrestrial insect species. It just doesn’t make any sense. “Climatic oscillations” still seems like such a vague handwave that I would prefer to leave it out, it’s really not a meaningful hypothesis even as speculation.

We have deleted the column with the suggested events from Table 2.

1. I’m a bit confused by the authors’ refusal to include the species they have provided new sequence for in the main text. I do see where they have indicated the newly sequenced species in Table S1, but that’s pretty difficult to find unless you read that long table closely. They should be proud of generating new data, and the description of that new data needs to be in the main text. There also needs to be at least a cursory description of the findings of the new data collection in the results section. None of this would add more than a few very simple sentences.

Done. Lines 115-118.

Other comments:

1. Appendix C is pointless and should be removed. The purpose of the paper is to make information easily accessible to readers. Adding unnecessary appendices and supplementary materials places an undue burden on readers.

Done.

1. I commented on the use of the term “rogue taxa” and whether it was necessary to include its description if they chose not to remove them. I don’t know what rogue taxa are and many readers won’t either. The authors need to explain this further.

Done.

Lines 295-296, 534-536: The authors claim that B. pauloensis should be considered as two different species on either side of the Andes, which neatly fits their hypothesis that “Andes uplift” is responsible for the vicariance event between B. pauloensis and B. pullatus. However, they provide no justification for this other than a vague and uncited claim that “several taxonomists suspect” that they are different species. This is a scientific paper and claims need to be supported by evidence. If the authors can provide evidence for this then the hypothesis still makes no sense – clearly, “Andes uplift” did not separate these two species but rather the two cryptic B. pauloensis species.

The text was modified, and two citations were inserted.

Table 1: remove space between B. and Brachycephalus in line 5.

Done.

Figure 2: Please remove the legend, as no reader could possibly use this legend to distinguish between these colors and glean any information about the figure.

Done.

Figure 2/3: I appreciate that the numbers in Figure 3 have been explained but I don’t see any events indicated by corresponding numbers in Figure 2, so I still don’t see what information they give us.

Done.

Round 3

Reviewer 1 Report

I am pleased to say that in the current revision, the authors have addressed all of my comments in the manuscript itself. The manuscript is now, in my opinion, suitable for publication.