Genetic Differentiation of Budburst Timing in Fagus crenata Populations along a Spatial Gradient in Late Frost Timing in the Hakkoda Mountains, Northern Japan

Round 1

Reviewer 1 Report

I  recommend that you revise the title: replace “along variation” with “along a spatial gradient.” Also replace in the term “spatial variation” with “spatial gradient” in the abstract (line 11), introduction (lines 57, 74, 83), and discussion sections (lines 349, 427). Its usage in lines 88, 345 is fine. In addition, you refer to “topographic variations” repeatedly in this manuscript. I think you mean “microsite variation” which has a different meaning. Topographical variation can be at any scale but microsites refer to a small geographic area. The significance of the paper is that microsite conditions may be influencing the genetics of a wind-pollinated species which is unusual and unexpected. The extensive use of jargon is very confusing for the reader.  The introduction and discussion have a lot of lengthy, unclear sentences that need to be written more succinctly to make the point clear.  Tables and figures are generally well done.

It’s interesting that trees with late budburst generally only occur in the basin and are not found along the hillsides. Why don’t we find trees with late budburst on the hillsides?  Is there a clear advantage to early budbreak that makes trees more competitive than trees that burst later in the season?  This competitive advantage is more prevalent in the hillsides where late frosts are less common. 

I recommend you review some of the literature in white spruce (Picea glauca) in North America, for example, see Lu and Man 2011. Assessment of assisted migration effects on spring bud flush in white spruce (Picea glauca [Moench] Voss) seedlings. The Forestry Chronicle. https://doi.org/10.5558/tfc2011-029. White spruce also breaks bud very early in the spring and is highly differentiated. The high tree-tree variation generally masks differences among provenances. I suspect with a larger sample size you might find a similar result. I recognize that large sample sizes are much easier to acquire with conifers than large-seeded hardwood trees!  

Specific comments:

Lines 44-48. Run-on sentence needs to be revised and clarified.

L48-49. Who or how does it “help?”  Please revise to be more concise.

L55. Delete “due to local adaptation” - it is understood.

L55-58. Please revise sentence. I suggest “…along a spatial gradient with topographic variation….”

L63. Revise. Suggestion: “…due to its tendency to leaf-out early in the spring.”

L64 and 66 seem contradictory. “Previous studies have reported that this species exhibits genetic differentiation along latitudinal gradients for temperature sum of leaf out [18,19]. However, the relationship between genetic variation and late frost timing has not been studied in this species.” I think the first sentence should state that it “exhibits phenotypic variation” along a gradient.

L74. Delete “of species to spatial variation”

L83. Spatial “gradient” instead of “variation”

L89. Revise: suggestion “to what extent is this trait heritable.”

L154. Sampling protocol isn’t clear. You selected 10 masting trees from each of the five sites?

L163. Only 44 survived out of how many? Why was the mortality was so high?  It would be helpful to understand why your sample size was so limited, and what factors contributed to seedling mortality. This is important because if selection was imposed in the nursery then your sample may be biased towards a particular phenotype that survived.

L325. Some extraneous text appears at the bottom of this page and into lines 334-337 (should be deleted)

L346. Delete phrases “spatial variation” and “timing.” I think it’s more accurate to state that “late frost is an abiotic factor that imparts selection in budburst timing.”

L349-351. Please revise this sentence to be more succinct. Too many words and phrases with no clear point.

L351. You refer to “topographic variations” repeatedly in this manuscript. I think you mean “microsite variation” which is similar, but different.   

L356. Was the trait value for temperature negative or flat? It looks flat and there is no r2 provided to validate the direction of the correlation.

L360-63. I think this logic is a stretch and I don’t think your data truly supports this because of the very small sample size and limited geographic area that you sampled. 

L360. This is the first mention of phenotypic plasticity. This should be mentioned in the introduction and contrasted with other terminology.

L389. I think you’re confusing maternal effects with epigenetic effects. They are not the same. Epigenetic effects result from methylation of DNA. Maternal effects relate to the seed coat/seed size that is a function of the mother. They can be deeply confounded but it’s important to clarify which type of variation you think is important in this system.

L407-8. The term “genetically differentiated” is used throughout the manuscript but I think it is sometimes confused with the term “selection.” I’m also confused with the logic in this sentence “Qst values would be larger than the Fst value….”  You can’t compare the Fst from other studies with your Qst and draw this conclusion.  Values of Fst are highly variable depending on the markers used and other factors.  It is likely that the population is undergoing diversifying selection (also referred to as disruptive selection) because a variety of phenotypes are maintained in the population depending on the forces of selection (growth vs frost damage).  We see a similar phenomenon with white spruce, Picea glauca, in North America. 

L448-452. Long run-on sentence. Please shorten and clarify.  

L482. Replace “spatial variation” with “spatial gradient”

L484. Delete “to spatial variation.” Local adaptation relates to the timing of late fatal frosts, not the spatial variation.

L485. The term “late” is used twice. I would revise to improve readability. For example, the last fatal frost may be delayed in basins vs hillsides.

L490. This sentence is unclear and should be written more succinctly: “…whether genetic differentiation in phenotypic plasticity of budburst timing is involved in local adaptation to spatial variation of late frost timing.” 

Fig 3. You might put the r2 on each chart or in the caption since this is displaying the heritability. Can you include standard errors?  Each value is the average over 5 years – there should be variability to report. I would expect a lot of year-year variation since budburst if a function of temperature sum.

Table 4. Please add a column that is indicative of the sample size (#mothers, #saplings/mother)

Figure 4. Please add r2 (or whether the lines are positive or negative) to the caption or the figure.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The authors conducted budburst studies in various populations of Fagus crenata Blume, an endemic species in Japan. Such studies are relevant to determine the adaptability of the species. On the other hand, both the methodology and the discussion of the results require substantial revisions.

The title must be corrected. Have the authors done any genetic research of populations of this species and have justified the differences found as being due to genetic differences? Also, the title is stylistically inappropriate, it repeats the same word "timing" which could be changed to "budburst stage" or "budburst phase".

Keywords. The keywords almost completely repeat the words in the title. I would suggest changing to make it easier and faster for the reader to find the publication.

 Abstract. This section should also help to find the article to decide whether it is worth examining in detail. In this case, the abstract does not provide clear essential information about the conducted studies and their results. Even the first sentence is very unclear, with unnecessary explanations in parentheses.

Introduction. This section is very extensive. As many as 22 references were discussed. Detailed descriptions of the investigations should be avoided or substantially shortened in this section (p. 2, lines 86-86; 91-103). Such descriptions should be provided in the Methods section. I consider it is necessary to justify the innovativeness of the research or methods employed. I missed that in this section.

Materials and methods. Data processing and methods used are presented. I have a question about collecting of samples for research. How could the authors justify that 3 or 6 saplings are enough to assess the genetic variation?  The number and collection of samples essentially determine the correctness of the experiment.

Results. The obtained data are presented in 4 tables, 4 figures and supplements. Visual information in the manuscript, i.e., figures, tables provide the information to the reader without the body text. And again, the authors should significantly correct the style of the sentences and do their best to make the essential points understandable to other scientists as well.

Discussion. The discussion is the place in the manuscript where authors should discuss their research findings in the context of other studies. This section is overloaded with various information on results of this study (p. 11, lines 366-379; p. 12, lines 435-444) without discussion. So, this section should base the state-of-the-art of the manuscript and must be corrected also.

Conclusions. I missed the generalization and the significance of the research. I do not think it is necessary to cite other authors in this chapter (p. 13, line 487-488). The sentence (p. 13, lines 485-489) is appropriate in the discussion section. The main results should be presented here: relations between budburst and environmental conditions; heritability of budburst timing etc. Novelty and research significance?

 

Minor issues:

1.       The list of the references provided does not meet the requirements for authors.

2.       What a reference is indicated on p. 4, line 128?

3.       The first time a plant species is mentioned in the text, it is necessary to indicate the author, F. crenata Blume.

Author Response

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Reviewer 3 Report

The manuscript entitled “Genetic differentiation of budburst timing in Fagus crenata  populations along variation in late frost timing in the Hakkoda  Mountains, northern Japan” describes genetic differentiation in budburst timing of Fagus crenata populations along spatial variation in late frost regime.  The main objective of the manuscript is relevant to be considered in programs of conservation of this genus and to be extrapolated it to other forest species.

I have several comments for this manuscript:

In the abstract, the novelty of the manuscript should be emphasized more clearly, and the research methodology should also be briefly mentioned here.

In the introduction (66 line) Maybe similar studies have been done with other beech species or other genera?

108 line. It would be good to add about the eco-climatic conditions of this beech tree, the typical vegetation, the soil.

137 line. six populations are mentioned in the text and 5 in parentheses

The end of the line 325 and 334-337 lines you must delete.

In all places, where you write "Sugimoto and Ishida (2022)" write like this "Sugimoto and Ishida [  reference ]"

In the conclusions, highlight the benefits and perspectives of this research

 

Author Response

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Round 2

Reviewer 1 Report

Two few minor spelling/grammatical errors (below) but overall the revised manuscript is much improved and easier to understand.  

Line 86: delete "that"

Line 87: variation (not variations)

 

 

 

Author Response

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Reviewer 2 Report

The authors have considered the comments and suggestions and revised the article accordingly.

Author Response

Thank you for your valuable comments on our revised manuscript.

Reviewer 3 Report

The authors have taken into account the comments made. All that remains is to arrange the tables and figures to fit on the page and the work can be accepted

Author Response

Thank you for your valuable comments on our revised manuscript.

Round 3

Reviewer 1 Report

Manuscript is much improved.  Minor suggestions below. 

Line 35. Replace “through” with “imposed by.”

L47. Replace “long” with “extensive” or “high”

L49-50. I think the point this sentence is trying to make is that migration of pollen among sites tends to “undo” any isolation-induced differences among populations. I would suggest a minor change in wording: replace “generates” with “sustains” high genetic diversity of seed.  Delete “before natural selection” because it is implied.

L56-57. Delete “with the same altitudes in mountainous regions” – the point is made with the latter part of the sentence (inversions).

L69. “local” not “lacal”

L81-82. You would only elucidate mechanisms of frost tolerance in THIS hardwood species since you are only sampling one species.   

L234. At first use of heritability I would introduce it as “narrow sense heritability.”

L235. At first use of family, write it as “offspring (family)” so it’s understood that “family” refers to the half-sib progeny of a single mother.

L237. This equation is correct when the offspring are half sibs AND when you are using the value for a single parent (as opposed to the mid-point of two parents).  

L312. These values seem correct, but to help the reader with the origin of the heritability estimates please put “estimated as twice the slope from parent-offspring regressions” in parentheses after the first phrase “Heritability estimates.”

Fig 3. I don’t think you need to show the bottom graphs if the only difference between the top and lower graphs are the basin points. The black points (S sites) are the same on top and bottom graphs.   

L388. Please put “latest bud-break time” in parentheses after “largest value” to help the reader interpret the results.

L390. Instead of “the populations have substantial genetic diversity for traits” I would state it this way: “substantial genetic diversity for traits was evident within populations as well.”  It’s interesting that the H1 lacks an “upper tail” (trees with delayed budbreak) while B1 populations lack a lower tail (trees with early budbreak). These results may be due to the small sample sizes, especially of S9. But I agree that your results show a lot of diversity within each of the other populations and among the populations, mainly between S9 and B1.  

L443-445. I agree with this sentence – but I recommend that you add a clarification. The heritability estimates might be lower across a larger gradient due to our inability to sort out the confounding effects of environment (snowpack, shading, etc). Reduced heritability estimates are not necessarily because the trait is under less genetic control.  

L444. Replace “fields” with “studies”

Author Response

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