Review History
Basis of single-seed formation in chestnut: cytomorphological observations reveal ovule developmental patterns of Castanea henryi

While I retain reservations about the quality of the micrographs in figure 3, I think that your statements in the paper probably reflect your observations. Readers who wish to challenge your interpretations are welcome to. Thank you for submitting to PeerJ.

Thank you for submitting your revised version. I apologize for taking a bit of time to get the decision back to you.

One of the reviewers is fully satisfied. The other reviewer has a two notable concerns (in addition to various minor issues of wording that will be easy to fix).

The first concern is supplemental table 1, which contained the raw data. In the earlier round reviewers and me both found this table difficult to read. Your solution was to drop it. But that is not acceptable, you need to include raw data in the supplement. Instead, you have to work on making the table readable. Perhaps ask some of your colleagues to have a look and see if they can understand.

The second concern is with figure too. The images are far too small. The features are difficult to see and the annotation texts obscure the features of interest. For example, figure 3b is supposed to show a pollen tube breaking thru a synergid cell and releasing sperm. Another example: we are expected to see a nucleolus in figure 3p but in fact we can barely see the nucleus. None of this, or other things you mention, can be seen in the tiny images. Higher magnification images must be shown. One approach would be to break this into two figures, or even three, and double or more the area of each panel. Perhaps a better approach would be to couple these low-mag survey views with higher magnification images of the cells of interest. Perhaps a combination of those approaches would work.

As an aside, the panels in figure 4 and 5 are generally large enough although larger ones for figure 5 would be better.

As before, I also read your text carefully. Generally, your text is much improved. I found a smaller number of places where I edited for clarity or validity. In some places you confuse writing about what you observed with what is true. The first is a statement of fact and the second is an inference. I have uploaded a marked-up text. Please incorporate the changes shown, unless you have a specific reason not to do so. I included a few notes about minor issues; I will not repeat them here.

When you submit your revised version, include your responses to both reviewer 3 and to me in a single file (the response or rebuttal letter). Also include track changes for the both changes I have indicated in the marked-up text file when you use them and for anything that you introduce.

Feel free to contact me off-line if you have specific questions. I look forward to getting the revised text.

This paper has been much improved. Both of the reviewers are generally supportive and I agree. But both reviewers found issues, about which I also agree. I read the paper carefully and I need to point out that a few further issues must be clarified.

One important issue are the data shown in Figure 5. On its face, this figure makes no sense. The figure shows data for three different times (0-6 weeks, 7 weeks, and 7 - 9 weeks), giving the percentage of various kinds of ovules within a single ovary. But most of the percentages are exactly the same for the three times. That cannot be real data (the percentages might be similar but not exactly the same) because these are destructive samples. Something is wrong here. Please revise completely to present what in fact you measured. The revision must include information about how many ovaries were sampled at each stage and the variability among the ovaries in the given percentages. That is, present mean ± standard deviation (or some other relevant indication of the variation).

Also problematic, the data in figure 5 partially overlap that of Table 1, maybe. In general, it is not clear whether the images and data for pollinated material come from bagged or open pollination. This should be made clear throughout.

Another issue is the staging of observations and figures. For example, the way figure 7 is described, the text reads as though the results are for ovules that have been fertilized. But I don’t see how it is possible to know that. Instead, I think that this material was sectioned at week 9 or 10. In other words, based on timing it is “after fertilization”. But the ovule itself might never have been fertilized. To avoid this ambiguity, please use the time of sampling to stage the data (sections, observations, etc).

There are also a large number of small problems. Some of these are with English but others relate to the content. I have marked up the manuscript extensively, with edits and comments. (A few of these overlap with points raised by the reviewers). I am not going to repeat them here. But please download my marked up version and look at all of the suggested changes and comments. If you have specific questions you are welcome to email me off line ([email protected]). I’ll be happy to help.

Thank you for submitting your interesting study to Peer J. I apologize for the time that has elapsed so far in review. I hope at least you find the reviewers’ and my comments helpful.

All three reviewers found interesting material in your submission but all of them identified notable problems. I read your paper carefully and agree. Synthesizing reviewer concerns, I identify three broad themes. Note that in your resubmission, I expect you to submit a letter where you reply to all of the points raised in review. If there are those you disagree with, that’s fine, just explain why.

First, more information about the methods is needed. You do not describe how many samples were investigated. For each method (figure, table) you need to state carefully how many ovules were examined. You need to describe the pollination treatment much more carefully. At some places in the text you refer to manual pollination but others to natural “free” pollination. Did you do anatomy on both? Likewise, for non-pollinated material, apparently you placed a bag of some kind over the flower. You need to describe how all of this was done. I wonder if you have a control for the bagging: for example, were hand pollinated flowers also bagged? Bagging itself could be influential. You need to describe the scanning electron microscopy. How were the flowers handled to make Figure 2?

Second, the paper needs a better structure. One approach would be to present successful development first, starting from the formation of the megagametophyte through to the mature embryo. With that established, then show deviations from the normal. I think this would make your results easier to follow.

Third, is there really ovule abortion post fertilization? In Figure 2, the ovary clearly has a single dominant ovule on week 7 after pollination, which is when you claim fertilization happens. So for the ovary in Fig. 2, all of the abortion is evidently pre-fertilization (or at the same time as). How many of this type of observation did you make? And in Figure 5, which purports to show fertilized ovules, how do you know the structures being called “zygote” are actually zygotes rather than some unusual cell structures following on from the abortion of a mature egg sac?

It is important to distinguish days after pollination, which you can easily monitor, from days after fertilization, which you can only infer from careful observations.

Now, I would like to make a few points of my own.

When you present figures that you believe represent abnormal situations, be careful about what you are actually observing. I mentioned above the ambiguity about whether you are actually seeing fertilization or looking at samples that are staged as being fertilized (but not demonstrated as such). In other cases, you describe some of the figure panels in highly specific terms but it is not always clear from the actual images how you can be so specific. It is fine to refer to “atypical anatomical [or cytological] arrangement” without over-interpreting.

Be sure to refer to all figures. In the current submission, you have a Figure 7 and 8 but these are never referred to in the text. Likewise, you refer to supplementary figures 1 and 2 but these were not uploaded.


Although reviewer 3 felt that you have a good introduction and state the scientific question being asked, I disagree in part. In the two main paragraphs of your introduction, you certainly do a good job in addressing general issues surrounding ovule abortion. But then, specific issues for your paper are jammed into the last paragraph containing only four lines. The main part of your introduction might just as well be introducing an ecological or evolutionary study on ovule abortion. Instead, you need to introduce the reader to the specific question(s) that you are asking. I think you are asking at which stages do ovules abort in chestnut and possibly with what kind of cellular appearance. Presumably this has been studied in other species; maybe in some, all of the abortion happens before fertilization? Or all of it happens after? Maybe some kinds of structures are recognized in other species that undergo ovule abortion. These kinds of possibilities need to be introduced and relevant literature cited.

Figures 1 and 2 are a helpful attempt to overview fruit development in chestnut. But Fig 1 e - g, although interesting, are not helpful for the issues at hand. By contrast, I don’t understand the relation between Figure 1 c and b. Is c a later stage? Were the hairs and so on removed? Fig. 1d is almost impossible to see; the SEM seems dark and not clear and the many lines drawn over simply obscure the structure. Perhaps an image taken on a dissecting scope would be better? Note that it would probably better to have a second panel that is only diagram next to this image, rather than superimposing all of the annotation.

As one of the reviewers noted, your description of the inflorescence, flower, and fruit (the first section of the results) is quite difficult to follow. The terminology is confusing and I think possibly not entirely standardized. What might help here would be a general diagrammatic figure that would introduce the reader to the relevant stages and structures. And such a figure could establish the terms you are using. That way, even a term is not standard, at least readers will know what you mean.

As one of the reviewers did, I found the SEM images in Fig. 3 to be unconvincing. The samples look poorly prepared and I think the surface morphology is not particularly informative. Perhaps using a dissecting scope would be more appropriate? Or this figure could simply be deleted.

The images in figure 2 imply that only one ovule enlarges. Is this always true? If so that might indicate some kind of signal that tells the fruit: “OK, we just have the one good one, grow!” This seems interesting to follow up.

I noticed that you used an editing service. Your English is grammatical, which is important and good. But from a science (or content) standpoint, the language is frequently off. Reviewers call out some of these places. I will add a few more here. Line 175: “All ovules can not form seeds after successful fertilization.” Do you understand the difference between “can not” and “do not”? The latter (do not) is a statement of fact; whereas, the latter is a statement about potential. I don’t see how you can claim that they “can not” without a great deal more evidence. Line 189: “The zygote has the advantage…” Here advantage is not the correct word. I don’t know what you mean. Line 196: “karyotyped” means to count the chromosome number and characterize their lengths. That is not what you mean here. Line 203: The word “excessive” is a matter of judgement. Someone who drinks and gambles and parties all night can be accused of ‘excessive’ behavior. If you introduced a transgene that caused a plant to make dozens of additional ovules, those could be called excessive. But the ovule production in chestnut is presumably adaptive. Use a word like “unused” or “extra” that carries no judgment.

The above comments on scientific language make an important point about commercial editing services: Most of these can deal only with English grammar. This misses the level of scientific language. If at all possible, ask a colleague who is a native English speaker and works on plants to read and comment.

In the figure legends, please provide detail about whether the image is a plastic section or a paraffin section.