Monuments Unveiled: Genetic Characterization of Large Old Chestnut (Castanea sativa Mill.) Trees Using Comparative Nuclear and Chloroplast DNA Analysis

Round 1

Reviewer 1 Report

Some corrections and additions are required:

Lines 106-118: A note to the natural stands with reference to the supplement needs to be added.

Line 115: The tree names are to be written in italics.

Behind Table 1, the line numbering starts again.

Line 9: Add a space between value and unit (95 °C).

Line 22: … Shaw et al [37], and Piredda et al. [38].

Line 28: … were deposited … (space missing)

Line 42: ... [42]. The … (dot missing)

Line 59: … MEGA 5.2 [54] … (space missing)

Lines 72 -73: The tree names are to be written in italics.

Line 83: The value 4.54 does not match to those in Table 3.

Table 2: The last column does not end in a line. The line before the Mean values is missing.

Line 239: 57.9 m (decimal dot)

Lines 270-271: Replace (Lindenmayer and Laurance 2016) with [8].

Line 321: (H5 and H6)

Line 325:  … H9 and H10)

Line 345: The tree names are to be put in quotation marks.

In total an interesting manuscript.

Author Response

Our detailed answers (in bold) to the  referee are as follows:

Reviewer #1

Q1: Lines 106-118: A note to the natural stands with reference to the supplement needs to be added.

Answer :Line 132.We have added the reference (Table S1)

Q2: Line 115: The tree names are to be written in italics.

Answer :Line 140 We have wrote the name in italics

Q3: Behind Table 1, the line numbering starts again.

Answer : We have corrected

Q4:  Line 9: Add a space between value and unit (95 °C).

Line 152 We have corrected

Q5: Line 22: … Shaw et al [37], and Piredda et al. [38].

Answer : Line 165 We have corrected

Q6: Line 28: … were deposited … (space missing)

Answer : Line 170 we have corrected

Q7: Line 42: ... [42]. The … (dot missing)

 Answer : Line 184 corrected

 Q8: Line 59: … MEGA 5.2 [54] … (space missing)

Answer : Line 196 corrected

Q9:  Lines 72 -73: The tree names are to be written in italics.

 Answer : Line 206 corrected

Q10: Line 83: The value 4.54 does not match to those in Table 3.

Answer : Line 215  corrected in 4.53

Q11: Table 2: The last column does not end in a line. The line before the Mean values is missing.

Answer : We corrected table 2

Q12: Line 239: 57.9 m (decimal dot)

Answer : Line 333 corrected

Q13: Lines 270-271: Replace (Lindenmayer and Laurance 2016) with [8].

Answer : Line 322 Corrected

Q14: Line 321: (H5 and H6)

Answer : Line 398 corrected

Q15: Line 325:  … H9 and H10)

Answer : line  401 done

Q16:Line 345: The tree names are to be put in quotation marks.

Answer : Line  416 done

Reviewer 2 Report

In this manuscript, the aim of the authors is to characterize genetically old chestnuts trees with nuclear and chloroplastic DNA markers. The highlighted genetic variability could be used as a genetic resource for chestnuts conservation programs.

General remark:

The genetic characterization helps to better understand chestnuts history in Italy. The little downside that I would put in it is that I don’t understand exactly how this genetic characterization will help in breeding or plantation programs to prepare the forests with environmental changes.

Introduction:

L41: how could be used this genetic variability to identify endangered populations without ecophysiological characterization.

L50: how old tress help to locate the less impacted areas?

L55: what are the the markers of a local adaptation?

L84: How long does it take to do a breeding program with a chestnut tree?

Material and methods:

L112 and 113: why didn’t the authors take the same number of bud and leaf samples on all trees? And for some trees only leaves? How to be sure that the root sucker sampled belongs to the chosen tree?

L17: why were the chloroplastic DNA analysis done on a subset of 27 trees among 99? How did the authors choose these 27 trees?

Results:

L69: root sucker have not been sampled on all trees, so how could the authors know if they don’t minimize the number of grafted trees? Do the grafted trees have the same rootstock?

L70: ate Tuscany and Umbria regions with important chestnuts production?

L102: the second axis of PCoA explain 17.46% of the variance, it’s not clear for me how it separates genotypes from Tuscany from the others

Discussion:

L183: although giant trees faced past modifications of the climate, but aren’t the present modifications of the climate too fast to preserve their adaptability?

L199: why are the long living chestnuts trees found only in mountain areas?

L204: why do forest habitats play a role in forest conservation during periods of land exploitation?

L212: maybe mountain communities have forgotten these trees because they were too difficult to access, not because they have a great respect for nature

The paragraph explaining how to estimate chestnut tree age is too long.

L267: how the micro-refugia explain the genetic divergence?

L279: why is there genetic erosion in Sicilian gene pool?

L285: is the relegation of ancient trees in isolated spots linked to a less productivity?

L291: what do the authors propose to preserve this genetic diversity?

Figures:

Table 1: are the different trees very distant from each other?

Figure 2: black bars are too thin

Figure 3: why are plastid network done one only 47 trees?

Conclusion:

I would have liked that the authors explain how, based on this study of genetic diversity, they were going to be able to improve the resilience of trees, identify endangered populations and evaluate adaptative potential?

Author Response

Revie61wer#2

General remark:

The genetic characterization helps to better understand chestnuts history in Italy. The little downside that I would put in it is that I don’t understand exactly how this genetic characterization will help in breeding or plantation programs to prepare the forests with environmental changes.

 The genetic analysis of long lived trees can help to find individuals with genetic characteristics to cope adverse environmental conditions, this “plus” genotypes can be used for programs of germplasm conservation and management.

Introduction:

Q: L41: how could be used this genetic variability to identify endangered populations without ecophysiological characterization.

Answer: This is a general statement on the well-known important role of forest genetic resources, and the motivation in reported in the cited papers. Specifically, low genetic diversity is universally acknowledged as an indicator of ongoing evolutive bottlenecks and/or strong selective pressure. Populations or species with low genetic diversity are considered to have low adaptability/resilience to next global changes and are therefore threatened. European chestnut has shown a low genetic diversity, to date. The possibility to find out new sources of genetic variation (in old-lived individuals) may open the way to future studies to properly correlate mechanisms and processes of selection/adaptation, obviously involving ecophysiological characterization. Line  47 we have changed the sentence

Q2: L50: how old tress help to locate the less impacted areas?

Answer: Compared to the highly impacted (or vulnerable, in the case of cultivated species) forest ecosystems of today, and given their maintenance through centuries, we can consider “less impacted” the areas or the forest patches where old living trees still grow.

We have changed the sentence line 61

Q3:L55: what are the markers of a local adaptation?

Answer: of course, there are not (yet) markers available, although many studies are ongoing worldwide in this respect (through field trials, QTL studies, the Candidate Genes approach, etc.). However, the genetic characterization of these extraordinary individuals can reveal special traits that have gone lost in the current germplasm during the centuries, just by random cases or because of the human selection for productive traits. Populations’ richness in private alleles (Par, see Table 3) can be also considered as an estimation of genetic traits possibly linked to local adaptation.

Q4: L84: How long does it take to do a breeding program with a chestnut tree?

We have changed the sentence  Line 101

Material and methods:

Q5: L112 and 113: why didn’t the authors take the same number of bud and leaf samples on all trees? And for some trees only leaves?

The collection was conducted in different times and not all trees were in the same vegetative stage.  We tried to collect the areal part and suckers because, due the grafting practise, it is possible to find different genotypes in the basal or in areal part. In some cases we were not able to collect basal part due the absence of suckers. (see Table 1).

 Line 136-137 in MM were changed .  Results line 203 was changed

Q6: How to be sure that the root sucker sampled belongs to the chosen tree?

We collected root suckers growing in the basal part of each tree, the trees collected were isolated.

Q7: L17: why were the chloroplastic DNA analysis done on a subset of 27 trees among 99? How did the authors choose these 27 trees?

Answer: Before our work, no data have been ever produced on plastid DNA sequence variation in European chestnuts; the only background study was performed with plastid microsatellites on 38 populations across southern Europe (Fineschi et al. 2000, cited in the text) with results pointing towards a strikingly low genetic diversity (11 haplotypes) and the non-existence of a clear geographic structure. Despite the low numbers, the subset of trees we analysed was sufficient to test and select the most suitable marker, based on reliability and efficacy, and to provide an indicative picture of the (previously overlooked) large diversity existing in giant-old individuals (10 haplotypes, three lineages and interesting connections with known glacial micro-refugia identified). The Discussion and Conclusion sections state the necessity to expand the number of investigated large old trees and other regions in future works. The subset was selected in order to represent all the geographical areas and the most interesting individuals (based on size, presumed age and relevance for the local communities) and it includes ca. 30% of the sampled trees. A second objective was to compare these trees with the close-by natural populations.The Discussion and Conclusion sections state the necessity to expand the number of investigated large old trees and other regions in future works.

Line 159-160 we added a sentence to specify how the samples were selected

Results:

Q8: L69: root sucker have not been sampled on all trees, so how could the authors know if they don’t minimize the number of grafted trees? Do the grafted trees have the same rootstock?

Answer: To better explained we changed in MM ( line 136-137) and  Results ( line 203).

The rootstocks were  different,  we find clonal areal part (see table S2). For the statistical analysis we used only the 106 unique genotypes observed.

Q9: L70: are Tuscany and Umbria regions with important chestnuts production?

Answer: Yes the Umbria and Tuscany regions have important chestnut production.

We add a sentence line 341. and we explained the importance of the chestnut cultivation in the next paragraph.

Q10: L102: the second axis of PCoA explain 17.46% of the variance, it’s not clear for me how it separates genotypes from Tuscany from the others

 Answer: The over all variance observed is 27% (the X axis explained 10.46% and the Y axis  17.46%). We have changed the sentence line 227-229. Moreover this result is congruent with the structure analysis.

Discussion:

Q11: L183: although giant trees faced past modifications of the climate, but aren’t the present modifications of the climate too fast to preserve their adaptability?

Answer: we agree with the comment, but it is also true that there is no other way to cope with the current trend. There is plenty of scientific evidence showing that natural migration will not be sufficient, and man-assisted (artificial) migration could not be the best solution for highly complex (and yet only partly known) ecosystems such as forests. Alfaro et al. Forest Ecology and Management 2014, 333: 76-87

Q12: L199: why are the long living chestnuts trees found only in mountain areas?

Answer: it is reported in the text: chestnut naturally grows in mountain areas and several mountain systems in the Mediterranean area experienced a low human impact because of inaccessibility or poor exploitation abilities. Lines 301-302

Q13: L204: why do forest habitats play a role in forest conservation during periods of land exploitation?

Answer: Line 302 was changed. the sentence is referred to “mountain habitats” and not “forest habitats”. For the reasons stated above (and reported in the cited papers), “mountain” forests very often represent extraordinary sources of biodiversity (under the specific, ecological and genetic points of view) with less impact than forests living in more anthropic areas.

Q14: L212: maybe mountain communities have forgotten these trees because they were too difficult to access, not because they have a great respect for nature

Answer: we agree with this comment: all the possible explanations are reported in the text

Line 314-315 “However, we cannot exclude that these plants simply survived because located in abandoned or in difficult-to-access sites or….”

Q15: The paragraph explaining how to estimate chestnut tree age is too long.

Answer:  Line 324 the sentence was shortened

Q16: L267: how the micro-refugia explain the genetic divergence?

Answer: glacial refugia are characterized by unique genetic signatures that have locally survived, remained isolated, and (possibly) differentiated from the others through the ages.

We have changed the sentence see line 355-356

Q17: L279: why is there genetic erosion in Sicilian gene pool?

Answer: The old trees from Sicily showed the lowest levels of genetic diversity, especially of Ar and PAr (Table 3), this parameter are generally considered the best indicators for the genetic diversity of populations.  However  we agree that this sentence could be confused, so we changed line 365-366

Q18: L285: is the relegation of ancient trees in isolated spots linked to a less productivity?

We collected ancient trees in different situation, or in very old orchard sites, or in isolated sites, in some cases near little village or few isolated house. We think that it is difficult to link their longevity with their productivity.

Q18 : L291: what do the authors propose to preserve this genetic diversity?

 Answer: it is reported in the Discussion  Line 300 “The catalog should be therefore implemented, and specific field campaigns at a finer scale should be launched for a detailed and continuous update…”) and Conclusion Line 429 (“continued individual monitoring, preservation of the habitats where they still live, and germplasm collection for safeguarding their important genotypes, should be ensured. Future studies should focus on…”) sections.

Figures:

Q19: Table 1: are the different trees very distant from each other?

In Table 1 are reported the geographic coordinates, in general the trees are distant from each other;  in some cases as Nave and Cento Cavalli are in the same areas,  in  Umbria and Tuscany some chestnut are in the same orchard sites.

Q20: Figure 2: black bars are too thin

The figure has been changed

Q21: Figure 3: why are plastid network done one only 47 trees?

 Answer: the network was produced on the investigated dataset (47 trees) see MM line 157-158

Conclusion:

Q22: I would have liked that the authors explain how, based on this study of genetic diversity, they were going to be able to improve the resilience of trees, identify endangered populations and evaluate adaptative potential?

Answer:

Line 422- 426 we modified the sentence

This is a genetic diversity study, aimed at a first description of relevant but yet unknown extraordinary individuals, as clearly stated in the title and at the end of the Introduction section (objectives and main aim). Our results can serve to drive future studies hopefully aimed at “to improve the resilience of trees … and evaluate adaptative potential”. For what concerns to “identify endangered populations”, as reported all through the Introduction (and all the cited studies), genetic diversity studies can identify populations with depleted genetic diversity, that are worldwide acknowledged as highly threatened with extinction. Giant old trees have demonstrated to provide new sources of genetic diversity that could be used to increase the species’ genetic resources. Increased values of genetic diversity generally imply higher resilience of populations and species, and high adaptive potential is generally linked to high genetic diversity. Worldwide literature agrees on these fundamental issues.