Review Reports - Xylem and Phloem Formation Dynamics in <i>Quercus ilex</i> L. at a Dry Site in Southern Italy

Round 1

Reviewer 1 Report

A pleasure to have reviewed this manuscript on xylogenesis and phloemogenesis of Quercus ilex L. in Italy.

Well structured, novel, with successful results and discussion, especially regarding the formation of thyllos in recent wood and the presence of starch and its possible relationship with abiotic agents.

A few minor issues.

1.- I recommend completing the scientific names of the species that appear in the manuscript with the authors.

2.- Sampling was carried out every fifteen days, therefore, over a year, 24 incisions were made in the tree. Please indicate whether these incisions were made along the circumference and at the same height and separation between them.

3.- Was the sampling performed on the trunk or branches? Please indicate it.

Congratulations to the authors for the quality of the manuscript and for the inclusion of technical notes on the difficulties of extraction and preparation of samples in hardwood species such as Quercus ilex.

Author Response

A pleasure to have reviewed this manuscript on xylogenesis and phloemogenesis of Quercus ilex L. in Italy.

We thank this reviewer for appreciating our study.

I recommend completing the scientific names of the species that appear in the manuscript with the authors.

Done. We added the Author when scientific names of species were introduced for the first time in the text.

Sampling was carried out every fifteen days, therefore, over a year, 24 incisions were made in the tree. Please indicate whether these incisions were made along the circumference and at the same height and separation between them.

We added requested information in the Materials and Methods at lines: 151-153.

Was the sampling performed on the trunk or branches? Please indicate it.

We added requested information in the Materials and Methods at lines: 151-153.

We thank the reviewer for highlighting the importance of the technical notes.

Reviewer 2 Report

This is a work that deals with important and current issues. Forest ecosystems are under pressure from increasing climate change. Understanding how trees react to stressful conditions, including water scarcity, is the first step to the more effective protection of forest ecosystems.

In my opinion, the manuscript is well written. My remarks are limited to a few observations and I present them in a synthetic form.

Line 131:
50 m s.l.m (Italian: sul livello del mare) = 50 m above sea level (English: above sea level ASL).

Figure.1
Plant growth is influenced not only by the precipitation but also by the relative air humidity and wind speed. Figure 1 should be completed with data on air humidity ad wind speed, if available. This would give a more complete characterization of the stress factors and the possibility of a slightly broader discussion of the results.

Figure 2C.
Cross-section of Quercus ilex viewed under polarized light is too dark and not easy to read. The photo should be lightened a bit.

In the “Materials and Methods” chapter, I did not find information about how many oaks were sampled for testing (from table 1 I can guess that there were eight trees).

Table 1 shows a huge variability of the examined features - their individual character. This has not been fully explained (in “Results” chapter – line: 254-256 and in “Discussion” chapter – line: 321-324).
What were the biosocial position of individual trees and their health condition? Maybe these factors influenced the observed high variability.

Author Response

In my opinion, the manuscript is well written. My remarks are limited to a few observations and I present them in a synthetic form.

We are grateful to this reviewer for acknowledging the value of the knowledge obtained through this study.

Line 131:

50 m s.l.m (Italian: sul livello del mare) = 50 m above sea level (English: above sea level ASL).

Sorry for this mistake. We corrected the text. Line: 125

Figure.1

Plant growth is influenced not only by the precipitation but also by the relative air humidity and wind speed. Figure 1 should be completed with data on air humidity ad wind speed, if available. This would give a more complete characterization of the stress factors and the possibility of a slightly broader discussion of the results.

Unfortunately these data were not available at the site.

Figure 2C.

Cross-section of Quercus ilex viewed under polarized light is too dark and not easy to read. The photo should be lightened a bit.

The picture was improved and replaced in the text.

In the “Materials and Methods” chapter, I did not find information about how many oaks were sampled for testing (from table 1 I can guess that there were eight trees).

The reviewer is right and the trees are 8 as reported at line 150. However, also as requested by the other reviewer, we added some more information on sampling technique. Lines: 150-153.

What were the biosocial position of individual trees and their health condition? Maybe these factors influenced the observed high variability.

The high variability among wood increments between trees in the same year and between years within the same tree is typical of trees in the Mediterranean ecosystems. We added some details and considerations in methods, results and discussion. Lines: 150-153; 262-263; 328-332.

Reviewer 3 Report

This paper presents results from eight Quercus ilex (Holm oak) trees from 1 stand in Italy. Over the course of a year’s sampling, every 2 weeks microcores were collected from the trees and prepared into slides for microscope analysis. This paper reports interesting results, such as the timing during the year when secondary xylem and secondary phloem were produced. Another highlight of the paper is that in 2 of the 8 trees, no secondary xylem was formed during the year, however secondary phloem was formed throughout the year – and during dry periods the ratio of xylem to phloem production decreased.

Where the paper needs to improve is in the description of the methodology, which is often not described, and also more thought needs to be given to the choice of wording throughout with regards to the anatomy, I generally found some of the terminology used odd throughout. Each of the following 4 paragraphs will discuss these particular points, but they add up in such a way that I found the writing to be ‘sloppy’.

The methodology for determining Intra-annual density fluctuations (IADFs) is not defined, in fact IADFs are not mentioned in the materials and methods section at all; is an IADF determined based on the number of pixels versus background in a given microscope section, and then assuming the cell wall had a density of 1500 kg/m3? Or something else? Significant clarity is needed here. I’m also not able to grasp the significance of the IADFs in the paper, all of the trees that produced xylem during the year produced a total of 1 IADF, whereas the 2 trees that did not produce any xylem of course did not have a IADF (table 1).

Post-cambial cells is throughout used but never defined. Post-cambial cells is not listed in the IAWA List of Microscopic features for hardwood identification, and it’s not found in other reference books, such as Charles Beck’s An Introduction to Plant Structure and Development:

https://www.researchgate.net/publication/294088872_IAWA_List_of_Microcopie_Features_for_Hardwood_Identification

The [30] reference that the authors reference (written by some of the authors) defines that the post-cambial cells are enlarging earlywood tracheids, however it could also mean enlarging latewood tracheids in that case depending on the season. Post-cambial could also mean one of the two tissues, either xylem or phloem, which is another reason I find the term odd. I would suggest ‘immature xylem’ or just ‘xylem’, which is much more clear than post-cambial since it actually describes what it is and it is consistent with the literature. This change would add clarity throughout the paper, for example the current sentence on line 242, “After the first week of May, with the driest months July and August (Figure 1), no PC cells were formed till to the end of August (Figure 5).” Changing to ‘xylem’ makes this sentence read more clearly, “After the first week of May, with the driest months July and August (Figure 1), no xylem cells were formed till to the end of August (Figure 5).” Note that the authors use ‘wood’ in Figure 6 to describe Figure 6B but use “PC” to describe 6A.

The CC definition, cambial cells, would be better suited as VC, which is the vascular cambium; since there is also the cork cambium, changing to VC or vascular cambium adds clarity. Other examples are PI which the authors define as a phloem fibres cluster, which is only used once in Figure 5 and is not needed to be defined as an acronym. Same comment for QA, or quantitative anatomy, which the authors use 4 times, and each time it would be easier to read if they just wrote quantitative anatomy. DOY, or date of year, is never defined.

Another example of the lack of description is there where the cores were collected is not discussed in the methods section, for example were cores collected at different radius around the tree, or the same radius with just different changes in height? On L412 the authors write, “We also observed high variability in the automatically structure along the circumference of the stem…”, however this point is not described in the methods and results, for example circumference of the stem is not mentioned before this point in the paper. A follow question here is, is it possible one side of the tree wasn’t producing secondary xylem but other sides of the tree were?

While not a major change. I understand why the authors presented Figure 1 where they did, but as a reader I find myself referring to Figure 1 and Figure 5 back to back – I suggest they make Figure 1 appear later on in the paper, with only 1 figure such that the original Figure 1 is on the top panel, and the original Figure 5 is on the bottom panel. This way a reader can see the timing of the secondary xylem and phloem formation and better link it with temperature and precipitation. The alternative to this would be to have the two figures on the same page, but given formatting it may be easier to just combine these into a A and B figure. This is a minor change but I think would add to the overall readability of the paper.

Fixing the above issues will add significant clarity to the paper. Beyond these issues, the cross section figures are of high quality (beyond the acronym choices) and do a good job of getting the authors points across. The formatting of some of the figures is a little weird at this point, I would suggest that some of the figures be rotated 90 degrees upon final publication, for example Figure 7 will be relatively small if fitted in the page, whereas if it is rotated it can be larger and thus be more readable (alternatively making a 2 row plot of 3 columns would also maintain the figure quality).

I will also say that all of these issues are fixable, but they need to be fixed prior to publication.

Specific comments organized by section and line number

Abstract

L13 – Your methods section (and Figure 5) shows that sampling is every 2 weeks and not weekly

L16 – see comments in overview on post cambial cells, why use a term other than xylem?

L21 – see comments in overview on IADFs not being explained

L21 – Rewrite to something like: “The fewest xylem cells were produced during the dry late spring and summer period (likely due to the low water availability), while the…”

L23 – early and late phloem?

L29 – I generally don’t think this sentence is needed in the abstract, I think spending more time on the results/conclusion is more important than this sentence. See comments later on regarding this section.

Introduction

L38 – this sentence needs a reference

L41 – I think ‘changing climate’ reads better than ‘future climate’ and changing ‘adjusted’ to ‘adapted’

L42 – change to, “For example, in the case of forests in southern Italy…”

L47 – the authors interchangeably use Q. ilex and holm oak, I’d suggest sticking to one or the other throughout the paper

L52 – it would be useful to list some of the major co-occurring species of these forests

L62 – replace ‘annual rings’ with ‘tree rings’

L62 – “As opposed to most Quercus species…’

L89 – here the cambium is the vascular cambium, nowhere is ‘vascular’ mentioned and it should be referred to as the vascular cambium here

L101 – quantitative anatomy is not needed to be abbreviated, too many acronyms equals a paper of alphabet soup.

Methods

Clarity is needed throughout the methods to describe what was actually done – see overview.

L157 – Define what biweekly means, based on Figure 5 biweekly means every 2 weeks (note that biweekly is not precise since it can mean every 2 weeks, or twice a week).

Results

L230 – define DOY

L258 – Table 1 – some changes to the table headers. Tree Number, Number of IADFs (and add explanation in the table somewhere), does the non-collapsed phloem also refer to an increment? Move the IADF column to the 2^nd column, and add a ratio between phloem and xylem increment.

Discussion

L311 – a ring porous tree growing slowly will also be difficult to impossible to identify the annual rings since it will just produce earlywood.

L325 – unless I missed it, the paper report results on the number of vascular cambium cells, but not the actual width. Here the discussion is talking about observing variation in the actual width (distance vs counting).

L398 - 4.3 Technical notes to improve sample preparation of Q. ilex

I generally find that most of this would be better suited in the methods section, since so few references are cited here (only 2 references).

What would you recommend the future researcher do to avoid the very wide rays? Sample 2 cores at each sampling event in the likely case that one of the cores will be less than useful?

L405 – what does thorn mean in the context of wood anatomy?

L412 – see overview on the circumference sentence, “We also observed high variability in the automatically structure along the circumference of the stem…”,

Author Response

This paper presents results from eight Quercus ilex (Holm oak) trees from 1 stand in Italy. Over the course of a year’s sampling, every 2 weeks microcores were collected from the trees and prepared into slides for microscope analysis. This paper reports interesting results, such as the timing during the year when secondary xylem and secondary phloem were produced. Another highlight of the paper is that in 2 of the 8 trees, no secondary xylem was formed during the year, however secondary phloem was formed throughout the year – and during dry periods the ratio of xylem to phloem production decreased.

We thank the reviewer for his/her general comments and we confirm we improved the description of methodology and wording as pointed out in the specific comments.

The description of IADFs in Q. ilex is reported in the Introduction and related references are reported as well. However, we added a clarification in the methods as requested. We agree that it makes no sense to report that trees not forming xylem also did not show IADFs, so we deleted the value 0 from the table. Lines: 178-180; 273-275; table 1.

https://www.researchgate.net/publication/294088872_IAWA_List_of_Microcopie_Features_for_Hardwood_Identification

We are aware that Post-cambial cells are not reported in the IAWA List of Microscopic features for hardwood identification, and the term is not found in other reference books, such as Charles Beck’s An Introduction to Plant Structure and Development. The authors know very well this reference literature. The absence of Post-cambial cells in the IAWA List is due to the fact that they are not a trait diagnostic for wood identification. However, the term “Post-cambial cells” has become of common use in the literature dealing with xylogenesis over the last 10 years.

Please also note that in ref n. 30 (Balzano et al. 2018), the authors do not define Post-cambial cells as “…enlarging earlywood tracheids,…”. This fragment of sentence that the reviewer is reporting in his/her comment is a part of the abstract in which the authors are describing a specific result of what happens in Pinus pinea between January and May. In that specific case, post-cambial cells were evolving in early-wood-like tracheids. In the same paper [30], the authors report the definition of Post-cambial cells giving a complete description of the features to classify them based on several references (Čufar et al., 2011; de Luis et al., 2011a; De Micco et al., 2016b; Rossi etal., 2006).

Therefore, we believe changing the term PC cells with just “xylem cells” would create confusion because a xylem cell is a generic term that is not indicating the stage of differentiation, thus it would result in imprecision.

Regarding figure 6, we corrected the legend.

Although we agree with the reviewer that VC would be an appropriate term, we prefer the term CC because it is broadly used in xylogenesis literature. We checked the acronyms and corrected a typo at line 175 about phloem fibres cluster. PI only remains in the legend of Fig. 5 to explain the label in the picture. QA is removed as acronym throughout the manuscript, while DOY has been defined at the first appearance in the manuscript (line 228).

The overall sampling covered the whole tree circumference, so we exclude that one side of the tree wasn’t producing secondary xylem but other sides of the tree were producing wood. We added clarifications in the Materials and methods section. Lines: 151-153

Figure 1 is needed to follow the methods and moving it in the results would make difficult to follow the results. We prefer maintain this order of pictures that is in line with the general scheme of papers on xylogenesis.

We are grateful to the reviewer for acknowledging the quality of pictures. We tried to change the panel composition of the figure but we believe that the one we proposed in Fig. 7 is the more suitable to help the reader follow the dynamics of phloem formation. Our setting follows the general scheme of pictures showing xylogenesis or phloemogenesis in which pictures are shown facing with cambial zone aligned.

I will also say that all of these issues are fixable, but they need to be fixed prior to publication.

We thank the reviewer because we think that having addressed his/her concerns have improved the manuscript.

Moreover, we accepted all minor modifications he/she suggested as follows:

C: L13 – Your methods section (and Figure 5) shows that sampling is every 2 weeks and not weekly

R: Done

C: L16 – see comments in overview on post cambial cells, why use a term other than xylem?

R: Please refer to R3

C: L21 – see comments in overview on IADFs not being explained

R: Please refer to R2

C: L21 – Rewrite to something like: “The fewest xylem cells were produced during the dry late spring and summer period (likely due to the low water availability), while the…”

R: We rephrased the sentence. Lines 23-25

C: L23 – early and late phloem?

R: Done

C: L29 – I generally don’t think this sentence is needed in the abstract, I think spending more time on the results/conclusion is more important than this sentence. See comments later on regarding this section.

R: We kept this sentence that was particularly appreciated by the other reviewers. Lines 30-32

C: L38 – this sentence needs a reference

R: Done. Line 41.

C: L41 – I think ‘changing climate’ reads better than ‘future climate’ and changing ‘adjusted’ to ‘adapted’

R: Done. Line 43

C: L42 – change to, “For example, in the case of forests in southern Italy…”

R: Done. Line 44

C: L47 – the authors interchangeably use Q. ilex and holm oak, I’d suggest sticking to one or the other throughout the paper

R: Done: we kept Q. ilex

C: L52 – it would be useful to list some of the major co-occurring species of these forests

Done. Lines 52-53

C: L62 – replace ‘annual rings’ with ‘tree rings’

R: Done. Line 64

C: L62 – “As opposed to most Quercus species…’

R: Done. Line 61

C: L89 – here the cambium is the vascular cambium, nowhere is ‘vascular’ mentioned and it should be referred to as the vascular cambium here

R: Done. Line 86

C: L101 – quantitative anatomy is not needed to be abbreviated, too many acronyms equals a paper of alphabet soup.

R: we removed the acronym QA throughout the text

C: Clarity is needed throughout the methods to describe what was actually done – see overview.

R: We added clarifications.

C: L157 – Define what biweekly means, based on Figure 5 biweekly means every 2 weeks (note that biweekly is not precise since it can mean every 2 weeks, or twice a week).

R: Done. Line 150

C: L230 – define DOY

R: Done. Line 228

C: L258 – Table 1 – some changes to the table headers. Tree Number, Number of IADFs (and add explanation in the table somewhere), does the non-collapsed phloem also refer to an increment? Move the IADF column to the 2nd column, and add a ratio between phloem and xylem increment.

R: Done

C: L311 – a ring porous tree growing slowly will also be difficult to impossible to identify the annual rings since it will just produce earlywood.

R: We agree, but adding comments on slowly-growing ring porous oaks is out of the focus of this paper.

C: L325 – unless I missed it, the paper report results on the number of vascular cambium cells, but not the actual width. Here the discussion is talking about observing variation in the actual width (distance vs counting).

R: We corrected cell width into cell number since we were talking about cambium production. Lines 333, 337

C: L398 - 4.3 Technical notes to improve sample preparation of Q. ilex

I generally find that most of this would be better suited in the methods section, since so few references are cited here (only 2 references). What would you recommend the future researcher do to avoid the very wide rays? Sample 2 cores at each sampling event in the likely case that one of the cores will be less than useful?

R: Although methodological, this paragraph reports the output and final considerations of this study and therefore it would not be suitable as methodological description. We prefer keeping this paragraph at the end of the discussion also to be coherent with positive comments made by the other reviewers. Obviously, we cannot advice a method to avoid wide rays, but we suggested to use a bigger Trephor (cutter diameter > 2mm): this would allow obtaining a larger sample ensuring having enough analysable xylem beside the wide ray. Line 421-422.

C: L405 – what does thorn mean in the context of wood anatomy?

R: We agree with the reviewer that “thorn” has not a specific meaning in the context of wood anatomy. Indeed, it is clearly a typo. We meant “torn” as a descriptive, not specific term, to indicate scratched, damaged, broken, cracked tissue.

C: L412 – see overview on the circumference sentence, “We also observed high variability in the automatically structure along the circumference of the stem…”,

R: we suppose the reviewer is meaning “We also observed high variability in the anatomical structure along the circumference of the stem…”. We rephrased the sentence at lines 414-416.

Round 2

Reviewer 3 Report

A couple of follow up points.

On post-cambial cells – C3 and R3

I appreciate the authors responses and why they choose the particular language, however the term post-cambial is still not defined in the revised manuscript.

On Figure 7 – C7 and R3

Given the desire to present this figure sequentially from left to right, I’d suggest rotating this figure 90 degrees so that the size of the figure is larger on the page and thus more visible.

Author Response

A couple of follow up points.

On post-cambial cells – C3 and R3. I appreciate the authors responses and why they choose the particular language, however the term post-cambial is still not defined in the revised manuscript.

We thank the reviewer for having appreciated our responses. We added a definition and a reference to clarify the definition of post-cambial cells.

On Figure 7 – C7 and R3. Given the desire to present this figure sequentially from left to right, I’d suggest rotating this figure 90 degrees so that the size of the figure is larger on the page and thus more visible.

We thank the reviewer for his/her comment and suggestion to increase the size of the picture. However, we think that we have to keep the orientation of the image as it is. The orientation we applied is in agreement with the rules of IAWA (International Association of Wood Anatomists) and in agreement with the previous 6 figures in the paper. We are afraid that rotating the figure would be wrong and it could confuse the readers.

To obtain a larger picture, the 6 images could be arranged in 2 rows (2 x 3 images) but in such case the increasing width of the currently formed phloem would be less obvious as shown following. (Since we cannot upload the image directly in the system, we embedded it in the attached letter).

Author Response File: Author Response.pdf