To say that it is a free country, but to also say that another man is owned by you clashes. The country is not truly free is anyone is owned by another.

means "war fortress" or strong willed. The name is from William Wilberforce who Champaign against the slave trade in Britain.

Mr. Shelby was just explaining how trusting Tom was and how much he valued him and his Christian beliefs, but was still willing to treat him as property when he had nothing else to offer.

This scene shocks through the contrast between a child’s innocent fear and the extreme resolve it awakens in his mother. The question “you won’t let him get me?” reveals how the threat of being sold has invaded even the safety of sleep, underscoring slavery’s reach into the most intimate spaces of family life. This pure and genuine question awakens something in Eliza- a strength to continue the fight to escape and bring Harry to freedom no matter what. Stowe intensifies the emotional impact by showing maternal love as a source of physical power, framing motherhood as both sacred and revolutionary in the face of slavery’s violence.

Carlo’s role as George’s sole source of comfort humanizes George, while the master’s casual order to drown the dog exposes how easily affection and life are destroyed under slavery’s logic of ownership. The violent image of a animal, especially that of a dog that is typically seen as “man’s best friend(‘s) death—followed by George’s flogging for refusing to comply—forces readers to confront the system’s capacity to punish compassion itself.

There is nuance in Eliza's identity that causes her to lose many opportunities, first as a Black person born during slavery and second as a woman who at the time were believed to be only as valuable as the man they were in wedlock to. This instance in the story highlights how misogyny impacts the character's perspective on the female figures through a misguided prediction on how one would escape their enslavement.

A tragic quote that stresses how far American society has progressed since the harrowing period that was slavery ended. A person cannot be sold as though they are a piece of food or a house appliance and in today's day and age it is quite bizarre to speak about a person as such. However, in the setting that Eliza and Uncle Tom conversate in this is all too normalized and told as though it were a part of an average conversation that has no weight to it.

The importance of this description of Rachel and her treatment of the slaves cannot be underscored for the juxtaposition it provides between the torture slaves were typically forced to endure and the warm hospitality she instead offers them. It presents to the audiences that the privileged and those who have been enslaved can live harmoniously as equals and not be oppositions to one another.

In that most potent of speeches George claims a sense of human dignity and condemns the racist logic of slavery enforced by laws that deny him basic rights. He voices his deep personal losses: a broken family, being forcibly taken away from his wife, and his identity becoming a racial epithet that serves as a justification for cruelty. For George, pride and a willingness to escape from such chains, even at significant risk, reflect a refusal of the society’s devaluation of his personhood. This excerpt illustrates the harsh facts of life of the enslaved mixed-race and their bravery to stand up for their rights. That passage is necessary for grasping the thematic matter of identity and injustice, an internal battle of enslaved beings claiming their equal potential against such laws.

This moment is a powerful demonstration of the dehumanization and cruelty that lies central to the slave trade. Aged, sick and crippled, Aunt Hagar desperately clings to her son Albert, hoping they would be sold together. But the auctioneer and buyers regard their distress as irrelevant, driving them apart in a brutal manner. The fear the boy has that they are going to be separated is very touching. You could barely see how slavery turned family members into instruments of commerce, dismantling generations-old connections and communities. It’s an emotional condemnation of the system’s savagery. I chose this because the auction scene is one of the most gut-wrenching and effective examples of the inhumanity of slavery in the text. It makes clear in graphic detail how families are torn apart, illustrating how immoral the practice was.

Tom takes hope and defiance in his faith and selflessness, that is, he believes in what God has put into place to keep his wife and children safe, accepting himself in his own faith that he will meet the weight of it to take his duties without fear, taking charge of the children’s safety. The passage articulates the heartbreak of slavery in family life but also the resilience of faith and piety as an aspect of bondage and the human spirit held together by enslaved communities.

Lucy's psychological devastation in the grotesque mechanics of the commerce of slavery is comparable only to a shot straight through the heart. The devastation she feels from this gunshot wound is crippling, she can't function, but this is "lawful trade" that does not care about her emotions. A tragedy that the reader must grapple with about the reality of how the machinery of slavery destroys lives and traumatizes its victims.

Stowe is giving the reader an emotional tug on the desperation of material separation. Here, she's framing the desperation as something so visceral that you cannot as a reader of good consitution separate yourself from the feeling. Maternal love is a common draw from Stowe, and this moment serves to exemplify its emotional impact on the audience. She's tugging on the heart strings.

Mrs. Shelby's acknowledgement in "fool that I was" shows an emergence of clarity in the fog of self-deception that she has been under about the institution of slavery. The awakening of her morality is what Stowe is hoping her audience sees, and how Mrs. Shelby's faith had given her a device of morality to support slavery all these years, and now she's seeing its evil.

This line shows Mr. Shelby being apprehensive about selling a child, especially as it'd mean being taken away from his mother. Children being separated from their mothers during slavery was common.

This line reflects a conversation between Mr. Shelby and the trader on which slave to buy– as Mr. Shelby declined an offer due to not deeming the woman as "worth it".

George’s response calls out Mr. Wilson’s hypocrisy by comparing slavery to something the man himself would never accept for himself. Instead of obeying Mr. Wilson’s advice to be compliant and accept life as a slave, George asks him to put himself in his shoes, imagining a scenario in which Wilson is treated the same way and asks him whether he would try to escape rather than claim it was “God’s will.”

When both Shelbys' realize the truth, we see that Mrs. Shelby is relieved and thankful that Eliza has escaped, believing it will keep her safe. In contrast, Mr. Shelby reacts with shock and concern, showing that his feelings are opposite to his wife’s happiness. However, it seems that his worry is more about his business dealings. I believe despite this, his kind treatment of Tom and the other enslaved people suggests he may still be hoping for Harry and Eliza’s safety, even if he fears the consequences of their escape.

This passage marks the point where Tom’s separation from his family becomes permanent and unavoidable. Before this moment, the pain of his sale is emotional and anticipatory, but when Haley places shackles on Tom’s ankles, that loss becomes physical and public. The chains are unnecessary for control, as Mrs. Shelby points out, but they are used to assert ownership and authority in front of Tom’s family and the surrounding community. Stowe underscores the cruelty of this act through the crowd’s reaction, showing that even people accustomed to slavery understand the injustice of chaining a man who has shown no resistance. Tom’s calm submission further emphasizes the imbalance of power and reveals how slavery punishes obedience and goodness rather than wrongdoing.

This sentence reveals the master's jealousy and how threatened he feels by George’s confidence, skill, and intelligence. Seeing George’s accomplishments at the factory makes the master view him as more than just a worker, so he acts quickly to reduce George back to a piece of property by taking him home and forcing him into hard labor.

Mrs. Bird doesn’t let John hide behind “politics.” She basically says: it’s easy to defend this law when it’s just an idea, but what would you actually do if a freezing, hungry runaway showed up at your door? John can talk about the “greater good” in theory, but Mary drags it into real life, where it’s suddenly obvious how cruel it is. Stowe is showing that these arguments only work when you don’t have to look the suffering person in the face.

Haley’s wording shows how slavery turns morality into something that can be measured by usefulness. Religion is not respected as faith or belief, but treated as a trait that makes an enslaved person more reliable and more profitable. By calling religion “valuable,” Haley reveals how even spiritual life is absorbed into the logic of the market.

The underlying message of this segment is that if a slave were seem to enjoy the new place they may be working at, their white slave owners may choose to change that notion for them by making work hard. This is all purely an inference of course of the times.

The separation of families had a strong impact on the slaves and they struggled with the idea of never seeing them again. This segment also displays the kindness that Tom had.

This custom of Tom's shows his want to read the Bible and find passages that affected the way he felt. It was his way of finding the important segments of the Bible.

This scene does well to show us the apathy of Haley, who callously sells this woman's child right out from underneath her the moment she steps away. He is more preoccupied with the idea of her raising a fuss or making a scene than how much agony she's in, the hurt he has caused and the inhumanity of selling a living, breathing, ten month old child. In this he is seeing profit and damage control, nothing more and nothing less. It makes note of how desensitized he has become to the reactions of women who he does this to, and the relief he felt in seeing her shut down and cry in a calmer manner. Even describing it as an advantage. 

Such a powerful scene! One meant to represent the sheer force of will that a good mother contains, the lengths at which she would go to save her own child! Barefoot, blood staining the white ice underneath her feet. Mothers know pain, they know blood, and in Eliza's case the force of her desperation and determination would be rather reflective of all those like her during that time. To show how fiercely she loved her child, the same as any other mother of any skin color. She bleeds red, just as a white woman would. This scene stands strongly as a means of showing Eliza's bravery and humanity, and the image of her leaping from ice sheet to ice sheet is a profound one.

This is a really important narrative moment. It not only portrays Tom's devotion to his owner, but is resistance to leave the life that is in the now to the life that isn't guaranteed. In a more broad sense it portrays slavery and the harshness of it. It's the only life he's ever sadly known.

I find the neutrality of Mr. Shelby interesting, the way his lack of religion is used almost as an illusion to his inability to stand firm and protect those under his care. It makes note that he respects the beliefs of his wife certainly, but in the end that respect means nothing when backed into a corner, and religion doesn't play a part in his decisions as it does others.

This sentence really stood out to me because it shows that religion at the forefront, is used as a justification for slavery and the lack of " betrayal" regarding not fulfilling the task of Haley. it becomes an interesting center point in the narrative as the more things go along, other characters steadily rely on religion as a end to the means.

This is yet again another mention of religion within the novel. However this time, it serves as a moment narratively where Eliza encourages George to have faith in his religion. It presents a level of duality because prior in the text, religion. was presented as frame as to why a slave would fulfill the request.

Mr. Shelby’s claim that he is a “humane man” is immediately undercut by the reality of the situation: he is considering selling a child into slavery. Stowe uses this moment to expose the moral contradiction at the heart of “kind” slave holders—those who see themselves as compassionate while continuing to participate in an inhumane system. The word "thoughtfully" emphasizes his self-image as moral, even as his actions betray that belief. He may be more “humane” than other slave holders, but the word lacks meaning in this situation. It is simply a word to make himself feel better.

Stowe utilizes character Mrs. Shelby to portray the recognizing towards organized Christianity in which it often defends slavery rather than eliminate it, exposing the hypocrisy and confessional complacency of not reforming and continue to partake in slave ownership.

Another example of Stowe’s literary tactic in breaking the fourth wall to connect and persuade her audience.

Stowe demonstrates through dialogue how the the character Mr. Shelby self-justifies his decision to Mrs. Shelby through social principle rather than an internal morality thus deviating from Christianity indoctrination and leaning on social customs.

Stowe breaks the fourth wall to forewarn of the central character giving immediate proclamation of his heroism rather than waiting for readers to later discover as one of literary tactics.

Stowe uses motherhood to have the readers feel empathetic for Eliza. Stowe includes how broken Eliza feels by showing that child loss was one thing that broke her and made her unhappy.

Of the Author-Choice Workshops, this type most caught my attention as finishing stories is something I often feel anxious about. Writing fiction in general is something I have little experience in, and I think I could benefit from extra help coming up with proper endings.

As someone with little experience going into this reading, I would have appreciated an inclusion of what CRP stands for, as it would help me visualize how to reverse the steps better in my head.

I have never participated in any workshop before, but I really like the inclusion of artists being able to ask for specific feedback on their work. When I first thought of workshops, I admit I only thought of the workshop giving feedback without any form of control from the artist.

I like that this author takes time to slow down her writing to add vivid imagery for the reader. Becuase it adds more detail and takes her a longer time to get to the point, it feels like she is showing us firsthand that the techniques she is writing about are not limited to narratives alone.

I think that a reason I find odes to other writers' works in some of the stuff I read entertaining is becuase I know that had I not taken the time to slow down and read closer, I'd have never found them in the first place. This technique can add a lot of texture to writing, and I can't wait to try it on my own.

I think that as a newer reader, these types of beginnings always used to annoy me. It would be useful for setting up information and the setting, but I think this is the first time I have thought about it in a way that actually fuels a character or narrator

I enjoyed reading this example, and I feel like it gave me a clearer understanding of the chapter "Meanders." I like that Alison didn't just dive into the plot choices of the author, but singled out specific names and word choices that added a deeper meaning to the entire story.

As someone who likes to learn as much as I can about literature, this is the first time I have heard novels described this way. I think that the complexity that can be found in them is the reason I tend to gravitate towards them over poetry.

I appreciate a large part of this class so far being devoted to breaking the stigma that fiction should be written in a singular form. I have honestly been noticing this form much more often after our first reading on the subject. I like that it takes pressure off the students while also pushing them to think critically about others ways fiction can be written.

As a very visual person, I love thinking about reading this way. I also feel like it introduces a depth behind simply reading a work of fiction. Not only do you travel through the characters, but you also travel through the specific choices and thoughts of the author.

I really like the connection that this author made. It hooked the audience, and I found myself questioning how the story pertained to the overall message about writing.

This example shows how a loop variable acts like a running total: it starts at 0, updates each time a condition is met, and stores the final count after the loop ends. The code is especially clear because it combines iteration (for letter in "Mississippi") with a conditional check (if letter == "i"), which is a common beginner pattern. You could make it even stronger by noting that this same structure works for counting anything in a list or string, not just letters.

This passage clearly explains that recommendation algorithms decide what users see by applying programmed rules to rank content like posts, friends, and ads. It also hints at why these systems matter: they shape users’ attention and experience on social media, often influencing what people believe is popular or important. You could strengthen it by briefly noting that these algorithms are usually optimized for engagement, not necessarily for accuracy or well-being.

I like how very it flows very well and shows imagery with what's going to be talked about

?

Helps me write a good thesis

?

True

Shows key primary knownledge of how readers should see a lit text.

turning a sweet, innocent moment into something that feels wrong

"he was rarely home early enough" - hard sentence to hear coming from a younger boy

visual

If only it weren't 25+ years later (and the PowerShell folks hadn't regressed on Windows).

Since its inception at the turn of the last century, the community college has played contradictory roles. On the one hand, it has served as a democratizing element in American higher education, by enabling greater access to postsecondary study. Because of its lower cost and less selective admissions policies, the community college enrolls students who would not otherwise have attended college. To its students, the community college offers a variety of options: credit courses and degree programs that are transferable to four-year colleges, subbaccalaureate degrees and certification for a range of occupations, and noncredit adult and continuing education. these schools offer an afforable option to go to college and ttransfer to a four year University

Elite colleges are desired by vast majority of potential students, and they are flooded with applicants, yet represent only 3% of student population. Why do we want the unobtainable??

• comma.ai operates its own $5M data center in-office to handle model training, metrics, and data storage, avoiding the "cloud tax."
• The facility consumes approximately 450kW at peak; power costs in San Diego (over 40c/kWh) totaled over $540,000 in 2025.
• Cooling is achieved using pure outside air with dual 48” intake and exhaust fans, utilizing a PID loop to manage temperature and humidity.
• The compute cluster consists primarily of 600 GPUs across 75 "TinyBox Pro" machines built in-house for cost efficiency and easier repairability.
• Storage is handled by several racks of Dell R630/R730 servers with ~4PB of total SSD storage, favoring speed and random access over redundancy.
• The software stack is kept simple to ensure 99% uptime, utilizing Ubuntu (pxeboot), Salt for management, and "minikeyvalue" for distributed storage.
• By owning their hardware, comma.ai estimates they saved $20M+ compared to equivalent compute costs in a public cloud environment.

Hacker News Discussion

• Users discussed the spectrum of infrastructure, ranging from pure Cloud (low cap-ex, high op-ex) to colocation and on-prem (high cap-ex, high skill requirement).
• A primary concern raised was "brain drain"—on-prem setups can become "legacy debt" if the senior engineers who built the custom systems leave without documenting unwritten knowledge.
• Commenters noted that AWS and other cloud providers are incentivized to keep architectures complex (microservices, serverless) to increase billing, whereas on-prem encourages efficiency.
• There was a debate regarding "software freedom" and the "WhatsApp effect," where small, highly motivated teams can outperform massive corporations by using lean, self-hosted stacks.
• Some users highlighted that while AWS pricing is expected to rise due to hardware costs, the "Quality of Life" and managed services still justify the cost for many startups without comma's scale.

comma-ai #self-hosting #datacenter #hardware-engineering

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary:

The study provides a comprehensive overview of genome size variation in two related species of the genus Epidendrum, which appear to be homoploid, although their DNA content more closely corresponds to that of heteroploid species. While I have a few serious concerns regarding the data analysis, the study itself demonstrates a well-designed approach and offers a valuable comparison of different methods for genome size estimation. In particular, I would highlight the analysis of repetitive elements, which effectively explains the observed differences between the species. However, I encourage the authors to adopt a more critical perspective on the k-mer analysis and the potential pitfalls in data interpretation.

Major comments:

R1. p. 9: Genome size estimation via flow cytometry is an incorrect approach. The deviation is approximately 19% for E. anisatum and about 25% for E. marmoratum across three repeated measurements of the same tissue over three days? These values are far beyond the accepted standards of best practice for flow cytometry, which recommend a maximum deviation of 2-5% between repeated measurements of the same individual. Such variability indicates a systemic methodological issue or improper instrument calibration. Results with this level of inconsistency cannot be considered reliable estimates of genome size obtained by flow cytometry. If you provide the raw data, I can help identify the likely source of error, but as it stands, these results are not acceptable.

__A: __Thanks a lot for pointing out this issue. We have identified the source of the wide interval after consulting with the staff of LabNalCit. We originally used human peripheral blood mononuclear cells (PBMCs) as a reference to estimate the genome size (GS) of P. sativum and used the resulting range to estimate the GS of Epidendrum. We calculated P. sativum's GS using a wide human GS range of 6-7 Gb, which resulted in a wide range of P. sativum GS and, consequently, in a wide range of GS for our samples. Therefore, the wide range reported is not an issue with the instruments, but about the specifics of the analysis.

__We have done the following changes: __

1. Reducing the range we calculated of P. sativum's GS using a narrower human genome size range (6.41-6.51; Piovesan et al. 2019; DOI: 10.1186/s13104-019-4137-z), and using these intervals to calculate our sample's GS.
2. We have explained our procedure in the methods, changed our results as required, and included a supplementary table with cytometry data (Supplementary Data Table 1).
3. Human peripheral blood mononuclear cells (PBMCs) from healthy individuals were used as a standard laboratory reference to calculate the P. sativum genome size. Pisum sativum and the Epidendrum samples were analyzed in a CytoFLEX S flow cytometer (Beckman-Coulter), individually and in combination with the internal references (PBMCs and P. sativum, respectively). Cytometry data analysis was performed using FlowJo® v. 10 (https://www.flowjo.com/). A genome size value for the Epidendrum samples was calculated as the average of the minimum and maximum 1C/2C values obtained from three replicates of the DNA content histograms of each tissue sample. Minimum and maximum values come from the interval of P. sativum estimations based on the human genome size range (human genome size range: 6.41-6.51; Piovesan et al. 2019).
4. The 1C value in gigabases (Gb; calculated from mass in pg) of E. anisatum ranged from 2.55 to 2.62 Gb (mean 1C value = 2.59 Gb) and that of E. marmoratum from 1.11 to 1.18 Gb (mean 1C value = 1.13 Gb; Supplementary Data Table S1).
5. We also eliminated from Figure 3 the range we had estimated previously.
6. Finally, we changed the focus of the comparison and discussion of the evaluation of the bioinformatic estimations, highlighting this deviation rather than whether the GS bioinformatic estimations fall within the cytometric interval. We calculated the Mean Absolute Deviation (MAD) as the absolute difference between the genome size estimates using k-mers and flow cytometry. This meant changing the results in P. 11 and 12 and adding to Fig. 3 two boxplots depicting the MAD. We have also added Supplementary Data Fig. S3 depicting the absolute deviations for E. anisatum and E. marmoratum per tool using the estimates generated from a k-mer counting with a maximum k-mer coverage value of 10,000 using 16 different values of k; a Supplementary Data Figure S5 depicting the mean absolute deviations resulting from the different subsampled simulated depths of coverage of 5×, 10×, 20×, 30×, and 40×; and finally a Supplementary Data Fig. S6 depicting the MAD changes as a function of depth of coverage for E. anisatum and E. marmoratum.

R1. p. 14 and some parts of Introduction: It may seem unusual, to say the least, to question genome size estimation in orchids using flow cytometry, given that this group is well known for extensive endoreplication. However, what effect does this phenomenon have on genome size analyses based on k-mers, or on the correct interpretation of peaks in k-mer histograms? How can such analyses be reliably interpreted when most nuclei used for DNA extraction and sequencing likely originate from endoreplicated cells? I would have expected a more detailed discussion of this issue in light of your results, particularly regarding the substantial variation in genome size estimates across different k-mer analysis settings. Could endoreplication be a contributing factor?

A:

We reworded the introduction p.3, 2nd paragraph to make our point on the effect of endoreplication on flow cytometry clearer. We eliminated the following sentence from discussion p. 15 : "Difficulties for cytometric estimation of genome size can thus be taxon-specific. Therefore, cross-validating flow cytometry and bioinformatics results can be the most effective method for estimating plant genome size, especially when only tissues suspected to show significant endoreplication, such as leaves, are available" We added the following, p. 18: Genome size estimation for non-model species is considered a highly standardized approach. However, tissue availability and intrinsic genome characteristics (large genomes, polyploidy, endoreplication, and the proportion of repetitive DNA) can still preclude genome size estimation (e.g. Kim et al. 2025) using cytometry and bioinformatic tools. Cross-validating flow cytometry and bioinformatics results might be particularly useful in those cases. For example, when only tissues suspected of showing significant conventional endoreplication, such as leaves, are available, bioinformatic tools can help to confirm that the first peak in cytometry histograms corresponds to 2C. Conversely, bioinformatic methods can be hindered by partial endoreplication, which only flow cytometry can detect.

     4. We included a paragraph discussing the effect of CE and PE on bioinformatic GS estimation P. 17:

Besides ploidy level, heterozygosity, and the proportion of repetitive DNA, k-mer distribution can be modified by endoreplication. Since endoreplication of the whole genome (CE) produces genome copies (as in preparation for cell division, but nuclear and cell division do not occur ), we do not expect an effect on genome size estimates based on k-mer analyses. In contrast, PE alters coverage of a significant proportion of the genome, affecting k-mer distributions and genome size estimates (Piet et al., 2022). Species with PE might be challenging for k-mer-based methods of genome size estimation.

R1. You repeatedly refer to the experiment on genome size estimation using analyses with maximum k-mer coverage of 10,000 and 2 million, under different k values. However, I would like to see a comparison - such as a correlation analysis - that supports this experiment. The results and discussion sections refer to it extensively, yet no corresponding figure or analysis is presented.

A:

We had previously included the results of the analyses using different k-mer coverage in the Supplementary Data Figure S2. We have added, to formally compare the results using analyses with maximum k-mer coverage of 10,000 and 2 million, a Wilcoxon paired signed-rank test, which showed a significant difference, p. 12: The estimated genome sizes using a maximum count value of 10,000 were generally lower for all tools in both species compared to using a maximum count value of 2 million (median of 2M experiment genome size - median of 10K experiment genome size= 0.24 Gb). The estimated genome size of the 2 million experiment also tended to be closer to the flow cytometry genome size estimation with significantly lower MAD than the 10K experiment (Wilcoxon paired signed-rank test p = 0.0009). In the 10K experiment (Supplementary Data Figure S2; S3), the tool with the lowest MAD for E. anisatum was findGSE-het (0.546 Gb) and for E. marmoratum it was findGSE-hom (0.116 Gb).

 2. We have added a boxplot in the Supplementary Data Figure S3 depicting the mean absolute deviations using maximum k-mer coverage of 10,000 and 2 million compared to flow cytometry.

Minor comments:

R1. p. 3: You stated: "Flow cytometry is the gold standard for genome size estimation, but whole-genome endoreplication (also known as conventional endoreplication; CE) and strict partial endoreplication (SPE) can confound this method." How did you mean this? Endopolyploidy is quite common in plants and flow cytometry is an excellent tool how to detect it and how to select the proper nuclei fraction for genome size estimation (if you are aware of possible misinterpretation caused by using inappropriate tissue for analysis). The same can be applied for partial endoreplication in orchids (see e.g. Travnicek et al 2015). Moreover, the term "strict partial endoreplication" is outdated and is only used by Brown et al. In more recent studies, the term partial endoreplication is used (e.g. Chumova et al. 2021- 10.1111/tpj.15306 or Piet et al. 2022 - 10.1016/j.xplc.2022.100330).

A:

We have reworded the paragraph where we stated "Flow cytometry is the gold standard for genome size estimation", as in the answer to Major comment 2. Additionally, we highlighted in the discussion how, while FC is the gold standard for GS estimation, studying multiple alternatives to it may be important for cases in which live tissue is not available or is available only to a limited extent (i.e. only certain tissues), p. 18 We have changed the term "strict partial endoreplication" to partial endoreplication (PE).

R1. p. 5: "...both because of its outstanding taxic diversity..." There is no such thing as "taxic" diversity - perhaps you mean taxonomic diversity or species richness.

__A: __We have changed "taxic diversity" to "species diversity".

R1. p. 6: In description of flow cytometry you stated: "Young leaves of Pisum sativum (4.45

pg/1C; Doležel et al. 1998) and peripheral blood mononuclear cells (PBMCs) from healthy

individuals...". What does that mean? Did you really use blood cells? For what purpose?

A: Please find the explanation and the modifications we've made in the answer to major comment 1.

R1. p. 7: What do you mean by this statement "...reference of low-copy nuclear genes for each species..."? As far as I know, the Granados-Mendoza study used the Angiosperm v.1 probe set, so did you use that set of probes as reference?

__A: __We rewrote: "To estimate the allele frequencies, the filtered sequences were mapped to a

reference of low-copy nuclear genes for each species" to:

To estimate the allele frequencies, the filtered sequences were mapped to the Angiosperm v.1 low-copy nuclear gene set of each species.

R1. p. 7: Chromosome counts - there is a paragraph of methodology used for chromosome counting, but no results of this important part of the study.

A: We are including a supplementary figure (Supplementary Data Figure 7) with micrographs of the chromosomes of E. anisatum and E. marmoratum.

R1. p. 12: Depth of coverage used in repeatome analysis - why did you use different coverage for both species? Any explanation is needed.

A: To make explicit the fact that the depth of coverage is determined automatically by the analysis with no consideration for the amount of input reads, but only of the graph density and the amount of RAM available (Box 3 in Novak et al. 2020), we rewrote:

"To estimate the proportion of repetitive DNA, the individual protocol analyzed reads corresponding to depths of coverage of 0.06× for Epidendrum anisatum and 0.43× for E. marmoratum." to

To estimate the proportion of repetitive DNA, the RepeatExplorer2 individual protocol determined a max number of analyzed reads (Nmax) corresponding to depths of coverage of 0.06x for Epidendrum anisatum and 0.43x for E. marmoratum.

R1. p. 16: The variation in genome size of orchids is even higher, as the highest known DNA amount has been estimated in Liparis purpureoviridis - 56.11 pg (Travnicek et al 2019 - doi: 10.1111/nph.15996)

A: We have updated it.

R1. Fig. 1 - Where is the standard peak on Fig. 1? You mention it explicitly on page 9 where you are talking about FCM histograms.

A: We reworded the results, eliminating the references to the standard internal reference.

Reviewer #1 (Significance (Required)):

Significance

This study provides a valuable contribution to understanding genome size variation in two Epidendrum species by combining flow cytometry, k-mer analysis, and repetitive element characterization. Its strength lies in the integrative approach and in demonstrating how repetitive elements can explain interspecific differences in DNA content. The work is among the first to directly compare flow cytometric and k-mer-based genome size estimates in orchids, extending current knowledge of genome evolution in this complex plant group. However, the study would benefit from a more critical discussion of the limitations and interpretative pitfalls of k-mer analysis and from addressing methodological inconsistencies in the cytometric data. The research will interest a specialized audience in plant genomics, cytogenetics, and genome evolution, particularly those studying non-model or highly endoreplicated species.

Field of expertise: plant cytogenetics, genome size evolution, orchid genomics.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary:

With this work, the authors provide genome profiling information on the Epidendrum genus. They performed low-coverage short read sequencing and analysis, as well as flow cytometry approaches to estimate genome size, and perform comparative analysis for these methods. They also used the WGS dataset to test different approaches and models for genome profiling, as well as repeat abundance estimation, empathising the importance of genome profiling to provide basic and comparative genomic information in our non-model study species. Results show that the two "closely-related" Epidendrum species analysed (E. marmoratum and E. anisatum) have different genome profiles, exhibiting a 2.3-fold genome size difference, mostly triggered by the expansion of repetitive elements in E. marmoratum, specially of Ty3-Gypsy LTR-retrotransposon and a 172 tandem repeat (satellite DNA).

Major comments:

Overall, the manuscript is well-written, the aim, results and methods are explained properly, and although I missed some information in the introduction, the paper structure is overall good, and it doesn't lack any important information. The quality of the analysis is also adequate and no further big experiments or analysis would be needed.

However, from my point of view, two main issues would need to be addressed:

__R2. __The methods section is properly detailed and well explained. However, the project data and scripts are not available at the figshare link provided, and the BioProject code provided is not found at SRA. This needs to be solved as soon as possible, as if they're not available for review reproducibility of the manuscript cannot be fully assessed.

__A: __We have made public the .histo files for all depths of coverage and cluster table files necessary to reproduce the results. We will also make public a fraction of the sequencing sufficient to reproduce our genome size and repetitive DNA results as soon as the manuscript is formally published. Whole dataset availability will be pending on the publication of the whole genome draft.

R2. The authors specify in the methods that 0.06x and 0.43x sequencing depths were used as inputs for the RE analysis of E. anisatum and E. marmoratum. I understand these are differences based on the data availability and genome size differences. However, they don't correspond to either of the recommendations from Novak et al (2020):

In the context of individual analysis: "The number of analyzed reads should correspond to 0.1-0.5× genome coverage. In the case of repeat-poor species, coverage can be increased up to 1.0-1.5×." Therefore, using 0.06x for E. anisatum should be justified, or at least addressed in the discussion.

Moreover, using such difference in coverage might affect any comparisons made using these results. Given that the amount of reads is not limiting in this case, why such specific coverages have been used should be discussed in detail.

In the context of comparative analysis: "Because different genomes are being analyzed simultaneously, the user must decide how they will be represented in the analyzed reads, choosing one of the following options. First, the number of reads analyzed from each genome will be adjusted to represent the same genome coverage. This option provides the same sensitivity of repeat detection for all analyzed samples and is therefore generally recommended; however, it requires that genome sizes of all analyzed species are known and that they do not substantially differ. In the case of large differences in genome sizes, too few reads may be analyzed from smaller genomes, especially if many species are analyzed simultaneously. A second option is to analyze the same number of reads from all samples, which will provide different depth of analysis in species differing in their genome sizes, and this fact should be considered when interpreting analysis results. Because each of these analysis setups has its advantages and drawbacks, it is a good idea to run both and cross-check their results."

Therefore, it should be confirmed how much it was used for this approach (as in the methods it is only specified how much it was used for the individual analysis), and why.

__A: __In Box 3, Novak et al (2020) explain that the number of analyzed reads (Nmax) is determined automatically by RepeatExplorer2, based on the graph density and available RAM. Therefore, the reported depths of coverage are results, not the input of the analysis. We tried different amounts of reads as input and got consistently similar results, so we kept the analysis using the whole dataset.

For the comparative analysis, we have added the resulting depth of coverage and explained that we used the same number of reads for both species.

Added to methods:

"For the comparative protocol, we used the same amount of reads for both species".

Added to results:

"To estimate the proportion of repetitive DNA, the RepeatExplorer2 individual protocol determined a maximum number of analyzed reads (Nmax) corresponding to depths of coverage of 0.06x for E. anisatum and 0.43x for E. marmoratum. "

"The RepeatExplorer2 comparative protocol determined a maximum number of analyzed reads (Nmax) corresponding to depths of coverage of approximately 0.14x for E. marmoratum and 0.06x for E. anisatum"

This is consistent with other works which utilize RepeatExplorer2, for example, Chumová et al (2021; https://doi.org/10.1111/tpj.15306), who wrote: "The final repeatome analysis for each species was done using a maximum number of reads representing between 0.049x and 1.389x of genome coverage."

Minor comments:

General comments:

• The concept of genome endoreplication and the problem it represents for C-value estimations needs to be better contextualised. It would be nice to have some background information in the introduction on how this is an issue (specially in Orchid species). Results shown are valuable and interesting but require a little more context on how frequent this is in plants, especially in Orchids, and across different tissues.

__A: __We have included information about the variation of conventional and partial endoreplication in plants.

Differences in CE may also occur between individuals or even respond to environmental factors (Barow 2006). In contrast, PE results in cells that replicate only a fraction (P) of the genome (Brown et al. 2017) and it has only been reported in Orchidaceae (Brown et al. 2017). CE and PE can occur in one or several endoreplication rounds, and different plant tissues may have different proportions of 2C, 4C, 8C ... nC or 2C, 4E, 8E, ... nE nuclear populations, respectively. The 2C nuclear population sometimes constitutes only a small fraction in differentiated somatic tissues and can be overlooked by cytometry (Trávníček et al. 2015). Using plant tissues with a high proportion of the 2C population (such as orchid ovaries and pollinaria) can help overcome this difficulty (Trávníček et al. 2015; Brown et al. 2017).

Comments and suggestions on the figures:

__R2. __In fig 1, the flow cytometry histograms need to be more self-explanatory. What are the Y axis "counts" of? Also, please either place the label for both rows or for each, but don't make it redundant. The axis fonts need to be made a bit larger too. If possible, explain briefly in the figure legend (and not only in the text) what each peak means.

__A: __We have modified the figure adding legends for Y and X axes, eliminated redundant labels, and changed the font size.

__R2. __Fig 5. Horizontal axis labels are illegible. Please make these larger (maybe make the plot wider by moving the plot legend to the top/bottom of the figure? - just a suggestion).

__A: __We consider the horizontal axis label to be superfluous and we removed it.

Small text editing suggestions:

R2. Methods, "Ploidy level estimation and chromosome counts" section. It would be easier for the reader if this paragraph were either divided into two methods sections, or into two paragraphs at least, since these are two very different approaches and provide slightly different data or information.

A: We slightly modified: "Chromosome number was counted from developing root tips" to

"Additionally, to confirm ploidy level, chromosome number was counted from developing root tips" and changed the subtitle to only "Ploidy level estimation".

R2. Methods, "Genome size estimation by k-mer analysis" section. Please specify whether the coverage simulations (of 5x to 40x) were made based on 1c or 2c of the genome size? I assumed haploid genome size but best to clarify.

A: We have added it to P7: "To assess the suitability of the whole dataset and estimate the minimum coverage required for genome size estimation, the depth of coverage of both datasets was calculated based on the flow cytometry 1C genome size values."

R2. Results, "Genome size estimation by k-mer analysis and ploidy estimation" section. In the first two paragraphs, the results presented appear to conform to anticipated patterns based on known properties of these types of datasets. Although this information confirms expected patterns, it does not provide new or biologically significant insights into the genomes analysed. It may be beneficial to further summarize these paragraphs so that the focus of this section can shift toward the comparison of methods and the biological interpretation of the genome profiles of Epidendrum.

__A: __We agree that those paragraphs deviate a little from the focus of our results. However, we believe they provide useful information both for pattern confirmation in a relatively understudied field and for readers which may not be very familiar with the methods utilized.

__R2. __Discussion, "Genome size estimation using flow cytometry" section. In the second paragraph, it is discussed how potential endoduplication events can "trick" the flow cytometry measurements. This has probably previously been discussed on other C-value calculation studies and would benefit from context from literature. How does this endoduplication really affect C-value measurements across plant taxa? I understand it is a well-known issue, so maybe add some references?

A: We have included in the Introduction information about CE and PE and their associated references. P. 3 and 4.

__R2. __Discussion, "Repetitive DNA composition in Epidendrum anisatum and E. marmoratum" section. In the second paragraph, when mentioning the relative abundance of Ty3-gypsy and Ty1-copia elements, it is also worth mentioning their differences in genomic distribution and the potential structural role of Ty3-gypsy elements.

A: We added this paragraph in P.20:

"Ty3-gypsy elements are frequently found in centromeric and pericentromeric regions, and may have an important structural role in heterochromatin (Jin et al. 2004; Neumann et al. 2011; Ma et al. 2023), particularly those with chromodomains in their structure (chromovirus, i.e. Tekay, CRM transposons; Neumann et al. 2011). Conversely, Ty1-copia elements tend to be more frequent in gene-rich regions (Wang et al. 2025A). However, Ty3-gypsy chromovirus elements can be found outside the heterochromatin regions (Neumann et al. 2011), and in Pennisetum purpureum (Poaceae) Ty1-copia elements are more common in pericentromeric regions (Yu et al. 2022)."

R2. Discussion, "Repetitive DNA composition in Epidendrum anisatum and E. marmoratum" section. In the third paragraph, it is mentioned that both species have 2n=40. I believe these are results from this work since there is a methods section for chromosome counting. This data should therefore go into results.

__A: __We have added the chromosome count micrographs as Supplementary Data Fig. S7

R2. Discussion, "Repetitive DNA composition in Epidendrum anisatum and E. marmoratum" section. I'd recommend expanding a bit more on repetitive DNA differences based on the RepeatExplorer results. Providing references on whether this has been found in other taxa would be helpful too. For example, Ogre bursts have been previously described in other species (e.g. legumes, Wang et al., 2025). Moreover, I consider worth highlighting and discussing other interesting differences found, such as the differences in unknown repeats (could be due to one species having "older" elements- too degraded to give any database hits- compared to the other), or Class II TE differences between species (and how these account less for genome size difference because of their size), etc.

A: We have rearranged and added discussion expanding on the role of repetitive DNA in E. anisatum and E. marmoratum and how it relates to the repetitive DNA in other species. This includes Ogre transposons, an expanded Ty1-copia vs. Ty3-gypsy discussion, and a section on unclassified repeats and can be found on P.19 to P.21.

Reviewer #2 (Significance (Required)):

Overall, this study provides a valuable contribution to our understanding of genome size diversity and repetitive DNA dynamics within Epidendrum, particularly through its combined use of low-coverage sequencing, flow cytometry, and comparative genome profiling. Its strongest aspects lie in the clear methodological framework and the integration of multiple complementary approaches, which together highlight substantial genome size divergence driven by repeat proliferation-an insight of clear relevance for orchid genomics and plant genome evolution more broadly.

While the work would benefit from improved data availability, additional contextualization of the problem of endoreduplication in flow cytometry, and clarification of some figure elements and methodological details, the study nonetheless advances the field by presenting new comparative genomic information for two understudied species and by evaluating different strategies for genome profiling in non-model taxa.

The primary audience will include researchers in non-model plant genomics, cytogenetics, and evolutionary biology, although the methodological comparisons may also be useful to a broader community working on genome characterization in diverse lineages. My expertise is in plant genomics, genome size evolution, and repetitive DNA biology; I am not a specialist in flow cytometry instrumentation or cytological methods, so my evaluation of those aspects is based on general familiarity rather than technical depth.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

A review on "Nuclear genome profiling of two Mexican orchids of the genus Epidendrum" by Alcalá-Gaxiola et al. submitted to ReviewCommons

The present manuscript presented genomic data for two endemic Maxican orchids: Epidendrum anisatum and E. marmoratum. Authors aim to determine the genome size and ploidy using traditional (flow cytometry and chromosome counts) and genomic techniques (k-mer analysis, heterozygosity), along with the repetitive DNA composition characterization.

Considering the genomic composition, the main difference observed in repeat composition between the two species was attributed to the presence of a 172 bp satDNA (AniS1) in E. anisatum, which represents about 11% of its genome but is virtually absent in E. marmoratum. The differences in the genomic proportion of AniS1 and Ty3-gypsy/Ogre lineage TEs between E. anisatum and E. marmoratum are suggested as potential drivers of the GS difference identified between the two species.

Our main concern are about the GS estimation and chromosome number determination. Along with many issues related to GS estimations by flow cytometry, results related to chromosome number determination are missing on the manuscript. Improvements in both techiniques and results are crucial since authors aim to compare different methods to GS and ploidy determination.

__R3. __Genome size: Following the abstract, it is no possible to understand that authors confirm the GS by flow cytometry - as clarified after on the manuscript. Please, since the approach used to obtain the results are crucial on this manuscript, make it clear on the abstract.

A: We have highlighted the congruence of flow cytometry and bioinformatic approaches in the abstract:

"Multiple depths of coverage, k values, and k-mer-based tools for genome size estimation were explored and contrasted with cytometry genome size estimations. Cytometry and k-mer analyses yielded a consistently higher genome size for E. anisatum (mean 1C genome size = 2.59 Gb) than * E. marmoratum* (mean 1C genome size = 1.13 Gb), which represents a 2.3-fold genome size difference."

__R3.__Flow cytometry methodology: For a standard protocol, it is mandatory to use, at least, three individuals, each one analyzed on triplicate. Is is also important to check the variation among measurements obtained from the same individual and the values obtained from different individuals. Such variation should be bellow 3%. The result should be the avarege C-value following the standard deviation, what inform us the variation among individuals and measurements.

__A: __We have done three technical replicates of each tissue of the individuals of E. anisatum and E. marmoratum. To show the variation from different replicates and tissues, we have included the Supplementary Data Table S1. Intraspecific variation on genome size is beyond the scope of this work.

__R3. __Checking Fig. 1, we could not see the Pisum peack. If authors performed an analysis with external standart, it should be clarified on Methods. I suggest always use internal standard.

Besides, comparing Fig. 1 for leave and pollinium, it seems to be necessary to set up the Flow Cytoemtry equipament. Note that the 2C peack change its position when comparing different graphs. The data could be placed more central on x-axis by setting the flow cytometry.

Action Required: Considering that authors want to compare indirect genomic approaches to determine the GS, I suggest authors improve the GS determination by Flow Cytometry.

Please, on Methodology section, keep both techniques focused on GS close one another. Follow the same order on Methodology, Results and Discussion sections.

__A: __We have made several changes on the estimation and reporting of the flow cytometry genome size estimation. Among these:

We have clarified the use of the P. sativum internal standard and PBMC's in methods (P.6). We have added the associated mean coefficient of variation for both the sample and the internal reference in Supplementary Data Table S1, in order to show that the variation is not the result of an instrument error. We have changed the order of the paragraphs in the methods section to follow the order in other sections.

__R3. __Chromosome count: In Introduction section (page 5), the authors explicitly aim to provide "bioinformatics ploidy level estimation and chromosome counting." Furthermore, the Methods section (page 7, subsection "Ploidy level estimation and chromosome counts") details a specific protocol for chromosome counting involving root tip pretreatment, fixation, and staining. However, no results regarding chromosome counting are presented in the manuscript. There are no micrographs of metaphase plates, no tables with counts, and no mention of the actual counts in the Results section or Supplementary Material. Despite this absence of evidence, the Discussion (Page 18) states: "ploidy and chromosome counts of both E. anisatum and E. marmoratum are the same (2n=40)." The value of 2n=40 is presented as a finding of this study, however, there is no reference to this results.

Action Required: The authors must resolve this discrepancy by either providing the missing empirical data (micrographs and counts). This detail needs to be reviewed with greater care and scientific integrity.

__A: __We have added the chromosome count micrographs as Supplementary Data Fig. S7.

Minor reviews (Suggestions):

__R3. __Refining the Title (Optional): Although the current title is descriptive, we believe it undersells the value of the manuscript. Since this study provides the first genome profiling and repeatome characterization for the genus Epidendrum and offers important insights into the calibration of bioinformatics tools and flow cytometry for repetitive genomes, I suggest modifying the title to reflect these aspects. The comparative access of GS is also an importante feature. This would make the article more attractive to a broader audience interested in genomics of non-model organisms.

__A: __We have changed the title to "Nuclear genome profiling of two species of Epidendrum (Orchidaceae): genome size, repeatome and ploidy"

__R3. __Botanical Nomenclature (Optional): Although citing taxonomic authorities is not strictly required in all fields of plant sciences, most botanical journals expect the full author citation at the first mention of each species. Including this information would improve the nomenclatural rigor of the manuscript and align it with common practices in botanical publishing.

A: We have added the citation of the taxonomic authorities:

"This study aims to use two closely related endemic Mexican species, Epidendrum anisatum Lex and Epidendrum marmoratum A. Rich. & Galeotti, to provide the first genomic profiling for this genus..."

__R3. __Abbreviation of Genus Names: I noticed inconsistencies in the abbreviation of scientific names throughout the manuscript. Standard scientific style dictates that the full genus name (Epidendrum) should be written out only at its first mention in the Abstract and again at the first mention in the main text. Thereafter, it should be abbreviated (e.g., E. anisatum, E. marmoratum), unless the name appears at the beginning of a sentence or if abbreviation would cause ambiguity with another genus. Please revise the text to apply this abbreviation consistently.

A: We have made the changes requested as necessary.

__R3. __Genome Size Notation: In the Abstract and throughout the text, genome size estimates are presented using the statistical symbol for the mean (x). While mathematically accurate, this notation is generic and does not immediately inform the reader about the biological nature of the DNA content (i.e., whether it refers to the gametic 1C or somatic 2C value). In plant cytometry literature, it is standard practice to explicitly label these values using C-value terminology to prevent ambiguity and eliminate the effect of the number of chromosome sets (Bennett & Leitch 2005; Greilhuber et al. 2005; Doležel et al. 2018). I strongly suggest replacing references to "x" with "1C" (e.g., changing "x = 2.58 Gb" to "mean 1C value = 2.58 Gb") to ensure immediate clarity and alignment with established conventions in the field.

__A: __We have revised the text in every instance, for example, in the results section:

"The 1C value in gigabases (Gb; calculated from mass in pg) of E. anisatum ranged from 2.55 to 2.62 Gb (mean 1C value = 2.59 Gb) and that of E. marmoratum from 1.11 to 1.18 Gb (mean 1C value = 1.13 Gb; Supplementary Data Table S1)."

__R3. __Justification of the Sequencing Method: Although the sequencing strategy is clearly described, the manuscript would benefit from a bit more contextualization regarding the choice of low-pass genome skimming. In the Introduction, a short justification of why this approach is suitable for estimating genome size, heterozygosity, and repeat composition, particularly in plants with large, repeat-rich genomes, would help readers better understand the methodological rationale. Likewise, in the Methods section, briefly outlining why the selected sequencing depth is appropriate, and how it aligns with previous studies using similar coverage levels, would strengthen the clarity of the methodological framework. These additions would make the rationale behind the sequencing approach more transparent and accessible to readers who may be less familiar with low-coverage genomic strategies.

__A: __We have added the following short sentence in P.7:

"This sequencing method produces suitable data sets without systematic biases, allowing the estimation of genome size and the proportion of repetitive DNA. "

__R3. __Wording Improvement Regarding RepeatExplorer2 Results: In the Results section, several sentences attribute biological outcomes to the RepeatExplorer2 "protocols" (e.g., "According to this protocol, both species have highly repetitive genomes..."; "The comparative protocol showed a 67% total repeat proportion, which falls between the estimated repeat proportions of the two species according to the results of the individual protocol"). Since the RepeatExplorer2 protocol itself only provides the analytical workflow and not species-specific results, this phrasing may be misleading.

A: We have rephrased these sections to emphasize that these are "the results of" the protocols and not the protocols themselves.

Reviewer #3 (Significance (Required)):

Significance

General assessment

Strengths

1.First Detailed Genomic Profile for the Genus Epidendrum: The study provides the first integrated dataset on genome size, ploidy, heterozygosity, and repeatome for species of the genus Epidendrum, a novel contribution for an extremely diverse and under-explored group in terms of cytogenomics.

Cross-validation of in vitro and in silico analyses: Flow cytometry is considered the gold standard for genome size (GS) estimation because it physically measures DNA quantity (Doležel et al. 2007; Śliwińska 2018). However, it typically requires fresh tissue, which is not always available. Conversely, k-mer analysis is a rapid bioinformatics technique utilizing sequencing data that does not rely on a reference genome. Nevertheless, it is frequently viewed with skepticism or distrust due to discrepancies with laboratory GS estimates (Pflug et al. 2020; Hesse 2023). In this study, by comparing computational results with flow cytometry data, the authors were able to validate the reliability of computational estimates for the investigated species. Since the 'true' GS was already established via flow cytometry, the authors used this value as a benchmark to test various software tools (GenomeScope, findGSE, CovEst) and parameters. This approach allowed for the identification of which tools perform best for complex genomes. For instance, they found that tools failing to account for heterozygosity (such as findGSE-hom) drastically overestimated the genome size of E. anisatum, whereas GenomeScope and findGSE-het (which account for heterozygosity) yielded results closer to the flow cytometry values. Thus, they demonstrated that this cross-validation is an effective method for estimating plant genome sizes with greater precision. This integrative approach is essential not only for defining GS but also for demonstrating how bioinformatics methods must be calibrated (particularly regarding depth of coverage and maximum k-mer coverage) to provide accurate data for non-model organisms when flow cytometry is not feasible.

Limitations

1. Limited Taxonomic Sampling: The study analyzes only two species of Epidendrum, which restricts the ability to make broad inferences regarding genome evolution across the genus. Given the outstanding diversity of Epidendrum (>1,800 species), the current sampling is insufficient to propose generalized evolutionary patterns. As the authors state by the end of the Discussion (page 18) "Future work should investigate to what extent LTR transposons and satellite DNA have been responsible for shaping genome size variation in different lineages of Epidendrum, analyzing a greater portion of its taxic diversity in an evolutionary context.". 2.Lack of Cytogenetic Results and Mapping: One of the major finding of this study is the identification of the AniS1 satellite as a potential key driver of the genome size difference between the species, occupying ~11% of the E. anisatum genome and virtually absent in E. marmoratum. While the authors use bioinformatic metrics (C and P indices) to infer a dispersed organization in the Discussion (Page 18), the study lacks physical validation via Fluorescence in situ Hybridization (FISH) - and a basic validation of the chromosome number. Without cytogenetic mapping, it is impossible to confirm the actual chromosomal distribution of this massive repetitive array, for instance, whether it has accumulated in specific heterochromatic blocks (e.g., centromeric or subtelomeric regions) or if it is genuinely interspersed along the chromosome arms. I suggest acknowledging this as a limitation in the Discussion, as the physical organization of such abundant repeats has significant implications for understanding the structural evolution of the species' chromosomes.

Advance

To the best of our knowledge, this study represents the first comprehensive genome profiling and repeatome characterization for any species of the genus Epidendrum. By integrating flow cytometry, k-mer-based approaches, and low-pass sequencing, the authors provide the first insights into the genomic architecture of Epidendrum, including quantitative assessments of transposable elements, lineage-specific satellite DNA, and repeat-driven genome expansion. This constitutes both a technical and a conceptual advance: technically, the study demonstrates the feasibility and limitations of combining in vitro and in silico methods for genome characterization in large, repeat-rich plant genomes; conceptually, it offers new evolutionary perspectives on how repetitive elements shape genome size divergence within a highly diverse orchid lineage. These results broaden the genomic knowledge base for Neotropical orchids and establish a foundational reference for future comparative, cytogenomic, and phylogenomic studies within Epidendrum and related groups.

Audience

This study will primarily interest a broad audience, including researchers in plant genomics, evolutionary biology, cytogenomics, and bioinformatics, especially those working with non-model plants or groups with large, repetitive genomes. It also holds relevance for scientists engaged in genome size evolution, repetitive DNA biology, and comparative genomics. Other researchers are likely to use this work as a methodological reference for genome profiling in non-model taxa, especially regarding the integration of flow cytometry and k-mer-based estimations and the challenges posed by highly repetitive genomes. The detailed repeatome characterization, including identification of lineage-specific satellites and retrotransposon dynamics, will support comparative genomic analyses, repeat evolution studies, and future cytogenetic validation (e.g., FISH experiments). Additionally, this dataset establishes a genomic baseline that can inform phylogenomic studies, species delimitation, and evolutionary inference within Epidendrum and related orchid groups.

Reviewer's Backgrounds

The review was prepared by two reviewers. Our expertise lies in evolution and biological diversity, with a focus on cytogenomic and genome size evolution. Among the projects in development, the cytogenomics evolution of Neotropical orchids is one of the main studies (also focused on Epidendrum). These areas shape my perspective in evaluating the evolutionary, cytogenomic, and biological implications of the study. However, we have limited expertise in methodologies related to k-mer-based genome profiling and heterozygosity modeling. Therefore, our evaluation does not deeply assess the technical validity of these analytical pipelines.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #3

Evidence, reproducibility and clarity

A review on "Nuclear genome profiling of two Mexican orchids of the genus Epidendrum" by Alcalá-Gaxiola et al. submitted to ReviewCommons

The present manuscript presented genomic data for two endemic Maxican orchids: Epidendrum anisatum and E. marmoratum. Authors aim to determine the genome size and ploidy using traditional (flow cytometry and chromosome counts) and genomic techniques (k-mer analysis, heterozygosity), along with the repetitive DNA composition characterization.

Considering the genomic composition, the main difference observed in repeat composition between the two species was attributed to the presence of a 172 bp satDNA (AniS1) in E. anisatum, which represents about 11% of its genome but is virtually absent in E. marmoratum. The differences in the genomic proportion of AniS1 and Ty3-gypsy/Ogre lineage TEs between E. anisatum and E. marmoratum are suggested as potential drivers of the GS difference identified between the two species.

Our main concern are about the GS estimation and chromosome number determination. Along with many issues related to GS estimations by flow cytometry, results related to chromosome number determination are missing on the manuscript. Improvements in both techiniques and results are crucial since authors aim to compare different methods to GS and ploidy determination.

Genome size: Following the abstract, it is no possible to understand that authors confirm the GS by flow cytometry - as clarified after on the manuscript. Please, since the approach used to obtain the results are crucial on this manuscript, make it clear on the abstract. Flow cytometry methodology: For a standart protocol, it is mandatory to use, at least, three individuals, each one analyzed on triplicate. Is is also important to check the variation among measurements obtained from the same individual and the values obtained from different individuals. Such variation should be bellow 3%. The result should be the avarege C-value following the standard deviation, what inform us the variation among individuals and measurements. Checking Fig. 1, we could not see the Pisum peack. If authors performed an analysis with external standart, it should be clarified on Methods. I suggest always use internal standard. Besides, comparing Fig. 1 for leave and pollinium, it seems to be necessary to set up the Flow Cytoemtry equipament. Note that the 2C peack change its position when comparing different graphs. The data could be placed more central on x-axis by setting the flow cytometry. Action Required: Considering that authors want to compare indirect genomic approaches to determine the GS, I suggest authors improve the GS determination by Flow Cytometry. Please, on Methodology section, keep both techniques focused on GS close one another. Follow the same order on Methodology, Results and Discussion sections.

Chromosome count: In Introduction section (page 5), the authors explicitly aim to provide "bioinformatics ploidy level estimation and chromosome counting." Furthermore, the Methods section (page 7, subsection "Ploidy level estimation and chromosome counts") details a specific protocol for chromosome counting involving root tip pretreatment, fixation, and staining. However, no results regarding chromosome counting are presented in the manuscript. There are no micrographs of metaphase plates, no tables with counts, and no mention of the actual counts in the Results section or Supplementary Material. Despite this absence of evidence, the Discussion (Page 18) states: "ploidy and chromosome counts of both E. anisatum and E. marmoratum are the same (2n=40)." The value of 2n=40 is presented as a finding of this study, however, there is no reference to this results. Action Required: The authors must resolve this discrepancy by either providing the missing empirical data (micrographs and counts). This detail needs to be reviewed with greater care and scientific integrity. Minor reviews (Sugestions): Refining the Title (Optional): Although the current title is descriptive, we believe it undersells the value of the manuscript. Since this study provides the first genome profiling and repeatome characterization for the genus Epidendrum and offers important insights into the calibration of bioinformatics tools and flow cytometry for repetitive genomes, I suggest modifying the title to reflect these aspects. The comparative access of GS is also an importante feature. This would make the article more attractive to a broader audience interested in genomics of non-model organisms. 

Botanical Nomenclature (Optional): Although citing taxonomic authorities is not strictly required in all fields of plant sciences, most botanical journals expect the full author citation at the first mention of each species. Including this information would improve the nomenclatural rigor of the manuscript and align it with common practices in botanical publishing.

Abbreviation of Genus Names: I noticed inconsistencies in the abbreviation of scientific names throughout the manuscript. Standard scientific style dictates that the full genus name (Epidendrum) should be written out only at its first mention in the Abstract and again at the first mention in the main text. Thereafter, it should be abbreviated (e.g., E. anisatum, E. marmoratum), unless the name appears at the beginning of a sentence or if abbreviation would cause ambiguity with another genus. Please revise the text to apply this abbreviation consistently.

Genome Size Notation: In the Abstract and throughout the text, genome size estimates are presented using the statistical symbol for the mean (x). While mathematically accurate, this notation is generic and does not immediately inform the reader about the biological nature of the DNA content (i.e., whether it refers to the gametic 1C or somatic 2C value). In plant cytometry literature, it is standard practice to explicitly label these values using C-value terminology to prevent ambiguity and eliminate the effect of the number of chromosome sets (Bennett & Leitch 2005; Greilhuber et al. 2005; Doležel et al. 2018). I strongly suggest replacing references to "x" with "1C" (e.g., changing "x = 2.58 Gb" to "mean 1C value = 2.58 Gb") to ensure immediate clarity and alignment with established conventions in the field.

Justification of the Sequencing Method: Although the sequencing strategy is clearly described, the manuscript would benefit from a bit more contextualization regarding the choice of low-pass genome skimming. In the Introduction, a short justification of why this approach is suitable for estimating genome size, heterozygosity, and repeat composition, particularly in plants with large, repeat-rich genomes, would help readers better understand the methodological rationale. Likewise, in the Methods section, briefly outlining why the selected sequencing depth is appropriate, and how it aligns with previous studies using similar coverage levels, would strengthen the clarity of the methodological framework. These additions would make the rationale behind the sequencing approach more transparent and accessible to readers who may be less familiar with low-coverage genomic strategies.

Wording Improvement Regarding RepeatExplorer2 Results: In the Results section, several sentences attribute biological outcomes to the RepeatExplorer2 "protocols" (e.g., "According to this protocol, both species have highly repetitive genomes..."; "The comparative protocol showed a 67% total repeat proportion, which falls between the estimated repeat proportions of the two species according to the results of the individual protocol"). Since the RepeatExplorer2 protocol itself only provides the analytical workflow and not species-specific results, this phrasing may be misleading.

Significance

General assessment

Strengths

1. First Detailed Genomic Profile for the Genus Epidendrum: The study provides the first integrated dataset on genome size, ploidy, heterozygosity, and repeatome for species of the genus Epidendrum, a novel contribution for an extremely diverse and under-explored group in terms of cytogenomics.
2. Cross-validation of in vitro and in silico analyses: Flow cytometry is considered the gold standard for genome size (GS) estimation because it physically measures DNA quantity (Doležel et al. 2007; Śliwińska 2018). However, it typically requires fresh tissue, which is not always available. Conversely, k-mer analysis is a rapid bioinformatics technique utilizing sequencing data that does not rely on a reference genome. Nevertheless, it is frequently viewed with skepticism or distrust due to discrepancies with laboratory GS estimates (Pflug et al. 2020; Hesse 2023). In this study, by comparing computational results with flow cytometry data, the authors were able to validate the reliability of computational estimates for the investigated species. Since the 'true' GS was already established via flow cytometry, the authors used this value as a benchmark to test various software tools (GenomeScope, findGSE, CovEst) and parameters. This approach allowed for the identification of which tools perform best for complex genomes. For instance, they found that tools failing to account for heterozygosity (such as findGSE-hom) drastically overestimated the genome size of E. anisatum, whereas GenomeScope and findGSE-het (which account for heterozygosity) yielded results closer to the flow cytometry values. Thus, they demonstrated that this cross-validation is an effective method for estimating plant genome sizes with greater precision. This integrative approach is essential not only for defining GS but also for demonstrating how bioinformatics methods must be calibrated (particularly regarding depth of coverage and maximum k-mer coverage) to provide accurate data for non-model organisms when flow cytometry is not feasible.

Limitations

1. Limited Taxonomic Sampling: The study analyzes only two species of Epidendrum, which restricts the ability to make broad inferences regarding genome evolution across the genus. Given the outstanding diversity of Epidendrum (>1,800 species), the current sampling is insufficient to propose generalized evolutionary patterns. As the authors state by the end of the Discussion (page 18) "Future work should investigate to what extent LTR transposons and satellite DNA have been responsible for shaping genome size variation in different lineages of Epidendrum, analyzing a greater portion of its taxic diversity in an evolutionary context.".
2. Lack of Cytogenetic Results and Mapping: One of the major finding of this study is the identification of the AniS1 satellite as a potential key driver of the genome size difference between the species, occupying ~11% of the E. anisatum genome and virtually absent in E. marmoratum. While the authors use bioinformatic metrics (C and P indices) to infer a dispersed organization in the Discussion (Page 18), the study lacks physical validation via Fluorescence in situ Hybridization (FISH) - and a basic validation of the chromosome number. Without cytogenetic mapping, it is impossible to confirm the actual chromosomal distribution of this massive repetitive array, for instance, whether it has accumulated in specific heterochromatic blocks (e.g., centromeric or subtelomeric regions) or if it is genuinely interspersed along the chromosome arms. I suggest acknowledging this as a limitation in the Discussion, as the physical organization of such abundant repeats has significant implications for understanding the structural evolution of the species' chromosomes.

Advance

To the best of our knowledge, this study represents the first comprehensive genome profiling and repeatome characterization for any species of the genus Epidendrum. By integrating flow cytometry, k-mer-based approaches, and low-pass sequencing, the authors provide the first insights into the genomic architecture of Epidendrum, including quantitative assessments of transposable elements, lineage-specific satellite DNA, and repeat-driven genome expansion. This constitutes both a technical and a conceptual advance: technically, the study demonstrates the feasibility and limitations of combining in vitro and in silico methods for genome characterization in large, repeat-rich plant genomes; conceptually, it offers new evolutionary perspectives on how repetitive elements shape genome size divergence within a highly diverse orchid lineage. These results broaden the genomic knowledge base for Neotropical orchids and establish a foundational reference for future comparative, cytogenomic, and phylogenomic studies within Epidendrum and related groups.

Audience

This study will primarily interest a broad audience, including researchers in plant genomics, evolutionary biology, cytogenomics, and bioinformatics, especially those working with non-model plants or groups with large, repetitive genomes. It also holds relevance for scientists engaged in genome size evolution, repetitive DNA biology, and comparative genomics. Other researchers are likely to use this work as a methodological reference for genome profiling in non-model taxa, especially regarding the integration of flow cytometry and k-mer-based estimations and the challenges posed by highly repetitive genomes. The detailed repeatome characterization, including identification of lineage-specific satellites and retrotransposon dynamics, will support comparative genomic analyses, repeat evolution studies, and future cytogenetic validation (e.g., FISH experiments). Additionally, this dataset establishes a genomic baseline that can inform phylogenomic studies, species delimitation, and evolutionary inference within Epidendrum and related orchid groups.

Reviewer's Backgrounds

The review was prepared by two reviewers. Our expertise lies in evolution and biological diversity, with a focus on cytogenomic and genome size evolution. Among the projects in development, the cytogenomics evolution of Neotropical orchids is one of the main studies (also focused on Epidendrum). These areas shape my perspective in evaluating the evolutionary, cytogenomic, and biological implications of the study. However, we have limited expertise in methodologies related to k-mer-based genome profiling and heterozygosity modeling. Therefore, our evaluation does not deeply assess the technical validity of these analytical pipelines.

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Referee #2

Evidence, reproducibility and clarity

Summary:

With this work, the authors provide genome profiling information on the Epidendrum genus. They performed low-coverage short read sequencing and analysis, as well as flow cytometry approaches to estimate genome size, and perform comparative analysis for these methods. They also used the WGS dataset to test different approaches and models for genome profiling, as well as repeat abundance estimation, empathising the importance of genome profiling to provide basic and comparative genomic information in our non-model study species. Results show that the two "closely-related" Epidendrum species analysed (E. marmoratum and E. anisatum) have different genome profiles, exhibiting a 2.3-fold genome size difference, mostly triggered by the expansion of repetitive elements in E. marmoratum, specially of Ty3-Gypsy LTR-retrotransposon and a 172 tandem repeat (satellite DNA).

Major comments:

Overall, the manuscript is well-written, the aim, results and methods are explained properly, and although I missed some information in the introduction, the paper structure is overall good, and it doesn't lack any important information. The quality of the analysis is also adequate and no further big experiments or analysis would be needed. However, from my point of view, two main issues would need to be addressed:

• The methods section is properly detailed and well explained. However, the project data and scripts are not available at the figshare link provided, and the BioProject code provided is not found at SRA. This needs to be solved as soon as possible, as if they're not available for review reproducibility of the manuscript cannot be fully assessed.
• The authors specify in the methods that 0.06x and 0.43x sequencing depths were used as inputs for the RE analysis of E. anisatum and E. marmoratum. I understand these are differences based on the data availability and genome size differences. However, they don't correspond to either of the recommendations from Novak et al (2020):

In the context of individual analysis: "The number of analyzed reads should correspond to 0.1-0.5× genome coverage. In the case of repeat-poor species, coverage can be increased up to 1.0-1.5×." Therefore, using 0.06x for E. anisatum should be justified, or at least addressed in the discussion. Moreover, using such difference in coverage might affect any comparisons made using these results. Given that the amount of reads is not limiting in this case, why such specific coverages have been used should be discussed in detail.

In the context of comparative analysis: "Because different genomes are being analyzed simultaneously, the user must decide how they will be represented in the analyzed reads, choosing one of the following options. First, the number of reads analyzed from each genome will be adjusted to represent the same genome coverage. This option provides the same sensitivity of repeat detection for all analyzed samples and is therefore generally recommended; however, it requires that genome sizes of all analyzed species are known and that they do not substantially differ. In the case of large differences in genome sizes, too few reads may be analyzed from smaller genomes, especially if many species are analyzed simultaneously. A second option is to analyze the same number of reads from all samples, which will provide different depth of analysis in species differing in their genome sizes, and this fact should be considered when interpreting analysis results. Because each of these analysis setups has its advantages and drawbacks, it is a good idea to run both and cross-check their results." Therefore, it should be confirmed how much it was used for this approach (as in the methods it is only specified how much it was used for the individual analysis), and why.

Minor comments:

General comments:

• The concept of genome endoreplication and the problem it represents for C-value estimations needs to be better contextualised. It would be nice to have some background information in the introduction on how this is an issue (specially in Orchid species). Results shown are valuable and interesting but require a little more context on how frequent this is in plants, especially in Orchids, and across different tissues.

Comments and suggestions on the figures:

• In fig 1, the flow cytometry histograms need to be more self-explanatory. What are the Y axis "counts" of? Also, please either place the label for both rows or for each, but don't make it redundant. The axis fonts need to be made a bit larger too. If possible, explain briefly in the figure legend (and not only in the text) what each peak means.
• Fig 5. Horizontal axis labels are illegible. Please make these larger (maybe make the plot wider by moving the plot legend to the top/bottom of the figure? - just a suggestion).

Small text editing suggestions:

• Methods, "Ploidy level estimation and chromosome counts" section. It would be easier for the reader if this paragraph was either divided into two methods sections, or into two paragraphs at least, since these are two very different approaches and provide slightly different data or information.
• Methods, "Genome size estimation by k-mer analysis" section. Please specify whether the coverage simulations (of 5x to 40x) were made based on 1c or 2c of the genome size? I assumed haploid genome size but best to clarify.
• Results, "Genome size estimation by k-mer analysis and ploidy estimation" section. In the first two paragraphs, the results presented appear to conform to anticipated patterns based on known properties of these types of datasets. Although this information confirms expected patterns, it does not provide new or biologically significant insights into the genomes analysed. It may be beneficial to further summarize these paragraphs so that the focus of this section can shift toward the comparison of methods and the biological interpretation of the genome profiles of Epidendrum.
• Discussion, "Genome size estimation using flow cytometry" section. In the second paragraph, it is discussed how potential endoduplication events can "trick" the flow cytometry measurements. This has probably previously been discussed on other C-value calculation studies and would benefit from context from literature. How does this endoduplication really affect C-value measurements across plant taxa? I understand it is a well-known issue, so maybe add some references?
• Discussion, "Repetitive DNA composition in Epidendrum anisatum and E. marmoratum" section. In the second paragraph, when mentioning the relative abundance of Ty3-gypsy and Ty1-copia elements, it is also worth mentioning their differences in genomic distribution and the potential structural role of Ty3-gypsy elements.
• Discussion, "Repetitive DNA composition in Epidendrum anisatum and E. marmoratum" section. In the third paragraph, it is mentioned that both species have 2n=40. I believe these are results from this work since there is a methods section for chromosome counting. This data should therefore go into results.
• Discussion, "Repetitive DNA composition in Epidendrum anisatum and E. marmoratum" section. I'd recommend expanding a bit more on repetitive DNA differences based on the RepeatExplorer results. Providing references on whether this has been found in other taxa would be helpful too. For example, Ogre bursts have been previously described in other species (e.g. legumes, Wang et al., 2025). Moreover, I consider worth highlighting and discussing other interesting differences found, such as the differences in unknown repeats (could be due to one species having "older" elements- too degraded to give any database hits- compared to the other), or Class II TE differences between species (and how these account less for genome size difference because of their size), etc.

Significance

Overall, this study provides a valuable contribution to our understanding of genome size diversity and repetitive DNA dynamics within Epidendrum, particularly through its combined use of low-coverage sequencing, flow cytometry, and comparative genome profiling. Its strongest aspects lie in the clear methodological framework and the integration of multiple complementary approaches, which together highlight substantial genome size divergence driven by repeat proliferation-an insight of clear relevance for orchid genomics and plant genome evolution more broadly.

While the work would benefit from improved data availability, additional contextualization of the problem of endoreduplication in flow cytometry, and clarification of some figure elements and methodological details, the study nonetheless advances the field by presenting new comparative genomic information for two understudied species and by evaluating different strategies for genome profiling in non-model taxa.

The primary audience will include researchers in non-model plant genomics, cytogenetics, and evolutionary biology, although the methodological comparisons may also be useful to a broader community working on genome characterization in diverse lineages. My expertise is in plant genomics, genome size evolution, and repetitive DNA biology; I am not a specialist in flow cytometry instrumentation or cytological methods, so my evaluation of those aspects is based on general familiarity rather than technical depth.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #1

Evidence, reproducibility and clarity

Summary:

The study provides a comprehensive overview of genome size variation in two related species of the genus Epidendrum, which appear to be homoploid, although their DNA content more closely corresponds to that of heteroploid species. While I have a few serious concerns regarding the data analysis, the study itself demonstrates a well-designed approach and offers a valuable comparison of different methods for genome size estimation. In particular, I would highlight the analysis of repetitive elements, which effectively explains the observed differences between the species. However, I encourage the authors to adopt a more critical perspective on the k-mer analysis and the potential pitfalls in data interpretation.

Major comments:

p. 9: Genome size estimation via flow cytometry is an incorrect approach. The deviation is approximately 19% for E. anisatum and about 25% for E. marmoratum across three repeated measurements of the same tissue over three days? These values are far beyond the accepted standards of best practice for flow cytometry, which recommend a maximum deviation of 2-5% between repeated measurements of the same individual. Such variability indicates a systemic methodological issue or improper instrument calibration. Results with this level of inconsistency cannot be considered reliable estimates of genome size obtained by flow cytometry. If you provide the raw data, I can help identify the likely source of error, but as it stands, these results are not acceptable.

p. 14 and some parts of Introduction: It may seem unusual, to say the least, to question genome size estimation in orchids using flow cytometry, given that this group is well known for extensive endoreplication. However, what effect does this phenomenon have on genome size analyses based on k-mers, or on the correct interpretation of peaks in k-mer histograms? How can such analyses be reliably interpreted when most nuclei used for DNA extraction and sequencing likely originate from endoreplicated cells? I would have expected a more detailed discussion of this issue in light of your results, particularly regarding the substantial variation in genome size estimates across different k-mer analysis settings. Could endoreplication be a contributing factor?

You repeatedly refer to the experiment on genome size estimation using analyses with maximum k-mer coverage of 10,000 and 2 million, under different k values. However, I would like to see a comparison - such as a correlation analysis - that supports this experiment. The results and discussion sections refer to it extensively, yet no corresponding figure or analysis is presented.

Minor comments:

p. 3: You stated: "Flow cytometry is the gold standard for genome size estimation, but whole-genome endoreplication (also known as conventional endoreplication; CE) and strict partial endoreplication (SPE) can confound this method." How did you mean this? Endopolyploidy is quite common in plants and flow cytometry is an excellent tool how to detect it and how to select the proper nuclei fraction for genome size estimation (if you are aware of possible misinterpretation caused by using inappropriate tissue for analysis). The same can be applied for partial endoreplication in orchids (see e.g. Travnicek et al 2015). Moreover, the term "strict partial endoreplication" is outdated and is only used by Brwon et al. In more recent studies, the term partial endoreplication is used (e.g. Chumova et al. 2021- 10.1111/tpj.15306 or Piet et al. 2022 - 10.1016/j.xplc.2022.100330).

p. 5: "...both because of its outstanding taxic diversity..." There is no such thing as "taxic" diversity - perhaps you mean taxonomic diversity or species richness.

p. 6: In description of flow cytometry you stated: "Young leaves of Pisum sativum (4.45 pg/1C; Doležel et al. 1998) and peripheral blood mononuclear cells (PBMCs) from healthy individuals...". What does that mean? Did you really use blood cells? For what purpose?

p. 7: What do you mean by this statement "...reference of low-copy nuclear genes for each species..."? As far as I know, the Granados-Mendoza study used the Angiosperm v.1 probe set, so did you use that set of probes as reference?

p. 7: Chromosome counts - there is a paragraph of methodology used for chromosome counting, but no results of this important part of the study.

p. 12: Depth of coverage used in repeatome analysis - why did you use different coverage for both species? Any explanation is needed.

p. 16: The variation in genome size of orchids is even higher, as the highest known DNA amount has been estimated in Liparis purpureoviridis - 56.11 pg (Travnicek et al 2019 - doi: 10.1111/nph.15996)

Fig. 1 - Where is the standard peak on Fig. 1? You mention it explicitly on page 9 where you are talking about FCM histograms.

Significance

This study provides a valuable contribution to understanding genome size variation in two Epidendrum species by combining flow cytometry, k-mer analysis, and repetitive element characterization. Its strength lies in the integrative approach and in demonstrating how repetitive elements can explain interspecific differences in DNA content. The work is among the first to directly compare flow cytometric and k-mer-based genome size estimates in orchids, extending current knowledge of genome evolution in this complex plant group. However, the study would benefit from a more critical discussion of the limitations and interpretative pitfalls of k-mer analysis and from addressing methodological inconsistencies in the cytometric data. The research will interest a specialized audience in plant genomics, cytogenetics, and genome evolution, particularly those studying non-model or highly endoreplicated species.

Field of expertise: plant cytogenetics, genome size evolution, orchid genomics.

It's interesting because when I was little. I though that Toyota, Honda, and Nissan where all Domestic American cars. Since there is a lot of them in the road. I now believe that Ford, GM, and Chrysler are having a hard time competing with them. Not to mention luxury vehicles because I see more Lexus than I do see Lincoln vehicles.

Also read other method

n Firefox you can clear cookies/cache for the current site via the padlock menu, and there’s also a keyboard shortcut to open the generic “Clear Recent History” dialog. For just this site (GitHub page)

Click the padlock icon to the left of the address bar.

Click Clear cookies and site data… (wording may be “Clear cookies and site data” for this site).

Confirm, then reload the page.​

That’s the fastest “per‑site” way and is usually what you want when debugging one page.

juliet finds out from the nurse that the man he just saw was romeo a montaque she is shocked and appalled bc her first love was someone their family hates

romeo goes and flirts with juliet and they engage in a kiss showing that they both are intrested in each other but the feast is ending and the guest are preparing to leave both are conflicted by their feelings and family relations to each other

capulet is giving a speech and welcoming the guest romeo spots juliet for the first time and is star struck by her beauty while tybalt notices romeo and is getting ready to fight him but he was stopped by capulet tybalt withdraws but vows he will get him

the capulet servants are running around preparing for the feast while joking and teasing each other

romeo tells mercutio about how he had a dream of bad things happening and is hesitant about going to the feast mercutio dissmisses him and goes into a rant about queen mab the dream fairy

romeoo benvolio and mercutioo is walking towards the feast romeo explains his unrequited love for rosaline and how hes too sad to enjoy himself and will only go as a observer

lady capulet and the nurse tells juliet it is almost her time for marriage and how a younge noble named paris is looking forward to marrying her and that she should observe him in the upcoming feast

the nurse is reminiscing about juliets childhood and how close they were

benvolio tells romeo t goo to the capulets feast to look at others by then would roosaline be less perfect romeo refuses but agrees to go to observe her beauty

benvolio advises romeo to stop the unrequited love for that woman romeo refuses saying hes still obsessed a servant wiith the list of people attending enters struggling to read the list romeo offers to help

paris ask for capulet permission to marry juliet but was denied bc juliet is too young and juliets consent matters too so he invites paris to a feast where juliet and other young girls would be present

benvolio ask romeo why he is so sad all the time for romeo to reveal that bc he is in love with a woman that doesnt love him back and benvolio try to cheer romeo up by saying there are other fishes in the sea and romeo said that the other fishes only remind him of the woman

the conversation shift too romeo and lady montaque is asking benvolioo where he is and is explained by montaque that he is always withdrawn and sad

the prince shows up and reprimand both houses

the fight have gotten so out of hand both lords of the houses comes in and are getting ready to join the battle

benvolio shows up to stop the fight but tybalt is looking for a fight so they engage in battle pulling the public into it

abraham confronts sampson for insulting the montaque and sampson dodges the question so abraham challenges sampson to a fight

they are insulting the montague and talkiing about how they will kill the maids and how they are ready to fight

the two servants samson and Gregory they are talking and joking about fighting the moontague and how they will violate the women

The phrases "loving hate" and "cold fire" are used as contradictions to show that Romeo's view of love is unstable.

The chaotic imagery further builds the sense of "mob mentality", showing how violence spreads uncontrollably

The Prince uses metaphor, comparing the men to beasts to emphasize how uncivilized they were being due to their rage.

This moment can potentially be foreshadowing.

Romeo utilizes a metaphor to portray love as something blinding and suffocating.

A small insult is enough to trigger a full fight, emphasizing how fragile the peace is in this setting.

Even servants are pulled into the situation, showing how deeply this fued controled society.

The violent/sexual language is utilized as a way to show the clear links to power and misogyny

biting the thumb (placing the thumb nail behind the upper teeth and flicking it forward) was a highly insulting gesture in 16th-century Italy and England.

What sets it off matters more than it seems. Watch Sampson shift control - he aims to pull the Montagues into motion before he moves. Blame must land elsewhere, never on him.

In Elizabethan cities, the person walking closest to the wall stayed out of the filth and sewage in the gutters

Claiming space by standing against the wall, Sampson asserts a higher position. His move reveals that the old conflict goes beyond open fights - dominance plays out through small acts each day. Public areas become quiet battlegrounds where status is tested without words.

The word final makes me think of death in this context but revising the final moments and looking back on them until it is offically final

Diction choice with the word cataloging. Makes me think of a magizine, like life is one big magazine full of documents that are put together. Usually people can keep or collect magizines too 

Personification again. The idea that the eye has a mind of its own and keeps track of things happening. Obsorbing information. 

Metaphorical document, not literal. Using the document as a metaphor for memory storage

The sun has a new role now and a new feeling. It takes on the role of a temporary mother, I think of the the sun hugging the baby as a sort of blanket.

Personification: the mind cannot literally file but it means the mind is saving the memory in the brain.

Contradiction between the cold of the winter and the warmth of the sun 

In the paragraph about inflation, why did government spending increase prices but not production in the 1970s, unlike in the 195?

Thatcher explains that Britain’s problems were hidden because most Western countries were becoming richer after the war. Even though Britain’s economy was growing, other countries were growing faster. This made Britain’s situation look better than it really was. When the economic boom of the post-war years slowed down in the 1970s, Britain’s weaknesses became clear.

... for people who have uvx installed.

Have etiquette with your classmates! Be polite.

pathos

CSR supports and guides a company’s efforts to achieve the triple bottom line by promoting responsible social and environmental practices alongside financial performance.

economic, social, and environmental

Apple provides a highly accessible section at the top of their homepage that lets users quickly find the devices they are looking for. Instead of relying on small text or complex menus, they use clear, easy-to-recognize images of each product, making it simple for users to identify items visually. This approach supports users who may have difficulty reading small text, are new to the brand, or may not remember the exact product names. By combining visual clarity with intuitive layout, Apple ensures that navigation is more perceivable, understandable, and user-friendly for a wider range of audiences.

Apple’s website presents its most important information inside clearly separated boxes and uses bold text to emphasize headings and key points. This creates clear visual groupings and hierarchy on the page. This is especially helpful for users with low vision, attention difficulties, or learning disabilities.

High Contrast & Distinguishable Colors Apple ensures good contrast between text and background for readability. High contrast improves visibility, making content readable for people with low vision or color differences.

The “Connect with a Specialist” link is clearly visible and consistently placed, allowing users to get human assistance through chat or guided support when navigating the site or making decisions. Helps users with cognitive disabilities, first-time users, or users who may feel overwhelmed by complex pages.

Clear, Structured Content Hierarchy The site uses clear semantic structures ) especially on multi-section pages so users and assistive tech like screen readers can easily understand and find content.

This code works well: tt <- if (!is.null(attr(Spread, "index"))) index(Spread) else time(Spread) y10 <- as.numeric(TB10YS) y3 <- as.numeric(TB3MS)

polygon( x = c(tt, rev(tt)), y = c(y10, rev(y3)), col = alpha("steelblue", 0.3), border = NA )

The impact of AI on the economy seems to boil down to this question: will companies and consumers favor human labor or the preservation of capital? The answer lies in the specific field AI is applied to.

This reflects how AI may not necessarily only eliminate certain jobs, it may also shift priorities to demand new skills that complement automation.

While the advancement of AI is tending towards full automation, it is suggested that human intermediaries are indispensable within the medical field as a result of bias towards human expertise.

When an occupation is automated by a system far superior to human computing ability, AI expansion can be justified. The loss of an individual or individuals' occupation may be justified by increased quality of care for those affected by serious illnesses.

The personalization aspect of AI is an avenue for innovation in many fields even beyond medicine, including education, finance, and even social networking.

This use of augmented labor could have significant social implications by expediting bureaucracy, an improvement that the US legal system could greatly benefit from.

This is a use-case in which the substitution of capital for labor is touted to increase efficiency. However, the consequences of this substitution may sacrifice the training of a younger generation of paralegals and neglect to prepare them for the industry that they hope to break in, as the entry-level job market will be oversaturated by robots.

When used correctly, AI can transform human decision making, taking the work of experts to a level that was previously unattainable and unequivocally improve the human condition. However, if too much trust is placed in AI prediction without the input of industry professionals, it can severely damage outcomes and credibility.

The effect of AI on the labor market is determined by advances in prediction technology--this affects current workplace values as it creates more avenues for capital to be directly substituted for labor. This will lead to changing workplace dynamics, resulting in the need for large portions of the workforce to adapt their skills to other fields, or gain new skills to succeed in another profession.

QUESTIONS:

What the hell is the answer to:

1) Exercise 2: In the editor, three vectors are defined. Each one represents the box office numbers from the first three Star Wars movies. The first element of each vector indicates the US box office revenue, the second element refers to the Non-US box office. In this exercise, you’ll combine all these figures into a single vector with name ‘box_office’. Next construct a ‘matrix star_wars’ with 2 rows and 3 columns.

2) Exercise 1: Write code to create an array ‘l’ with 3 sheets of 2 rows and 4 columns, filled with the first 5 letters of the alphabet.

The possibility to fill a matrix by rows is possibly the only advantage of matrix() over array().

reply to u/CaliKelli989 at https://old.reddit.com/r/typewriters/comments/1qx43wy/smith_corona_classic_12_for_75worth_it_for_my/ on signaling by online typewriter sales

Where you're selling is one of the biggest signals of all. Selling machines for over $250 on Facebook requires way more signaling on the part of a professional or semi-professional seller. Mr&Mrs are doing a whole lot more work on restoring their machines than the average "blow and go" level that Janet and her significant other are likely doing (or that done by the average shop), as a result they're doing more work to show that, but they're occupying a dramatically different market space. Who is offering warranties on their work? Who is recovering platens? Who is explicitly stating the quality of the rest of their rubber? (Note that Janet isn't saying anything about the rubber washers on her SM3s, nor did they say anything about the rubber feet or the feet on the cases. Were they all replaced?)

Most professional shops and restorers are selling via their physical shops or their own websites instead of eBay, which takes steep cuts, or FB where it's harder for their much better quality machines to stand out amidst similarly priced dirtier machines. (Most pros also refuse or prefer not to ship when they can avoid it, so online presence doesn't "buy" them much.) There's a huge gulf in the levels of work that Walid Saad or Lucas Dul are offering in complete tear downs and restorations and the simple clean, oil, and adjust operations that are being offered by average pro shops and that's different again from what I suspect Janet is probably offering. This doesn't even get into the space of the lowest level "flippers" and vintage/antique shops whose only value add is finding and offering machines. As a point of reference, Lucas is doing less than a full restoration a month in an average year. The rest is cleaning machines for straight sale and then repairs that walk in the door. I'd suspect that he doesn't have more than a dozen machines in stock that are ready for sale today compared to a multi-person operation like Typewriter Muse which has nearly 30 machines on the shelf ready to go.

There's a huge spectrum in the level of restorations being offered out there. Very few people appreciate any of the differences.

The issue is that many people starting out don't want to pay a lot for a clean/restored machine, so they're fine with something that "works". Generally they don't know what they're missing from a finely tuned machine. At the other end are serious collectors, who often have the knowledge and expertise to service their own machines. The biggest issue with the market is the huge gulf of information imbalance between the novice buyers and novice sellers and the professionals.

Hope this helps on the differentiation that's available out there...

I continued working on the inflation rate in the United States. The DF-GLS test:

summary(ur.ers(400 * diff(PCECTPI), model = "trend", lag.max = 2)).

The output:

Value of test-statistic is: -2.7078

Critical values of DF-GLS are: 1pct 5pct 10pct critical values -3.48 -2.89 -2.57

Having AI images of real children is not protecting children.

I do think that some people who are not aware of their pedophilic tendencies yet may be introduced to them with the use of AI pornography. Additionally, this could easily become a joke for teenagers to use for shock value.

Some people who engage in CSA are using technology that law enforcement cannot access, how could watermarks stop this?

People are uploading images of specific children into these AI platforms that create the content, therefore, there are still victims in need of help.

I do not think that a "craving" for pedophilic behavior and drugs is comparable. I do agree that the root of pedophilic behavior is biological, but that does not make it ok to engage in sexual acts involving children, that includes pornography.

While I agree that physically, Ai generated child sexual material may be less harmful, this is not true of mental and emotional effects. Additionally, that material has to be generated using actual images. If anything, this allows more children to be exploited.

In the last decade(s) we have had a lot of rights of these people vs rights of these people but little focus on firmness and efficiency?

Relatable and Yes, our choices will change the fate of humanity, but also they were already influenced by the choices made here in this doc. and so on. Free will and not. Important but only in combination with other people.

Considering "how do we protect our government and make sure it continues to protect our rights" is quite refreshing, when we've allowed this to decay, and indeed, "a government should be able to protect its existence" is often argued against when it comes to discussions regarding things like social media or infringing on certain economic rights

Builds up quick

Where is your proof and backing?

I'd be fuming too if it was that hot. (literally)

THE ULTIMATE ROAD RAGE STARTER!!!

So payment has to do with this subconsciously?

That targets my age range.

Dang that is CRAZY.

via Marty Morren https://www.facebook.com/groups/705152958470148/posts/1190871013231671/

This is such an awesome effort. Some thoughts while reading:

I have been wary of claims-based review given how hard it is to decouple claims from those made to chase editorial outcomes. I’d be very interested if we might prospectively use this type of result extraction to re-derive a supported claim (regardless of whether that was the claim made) and have that those be the content we operate on. I could even imagine authors using this type of supported content earlier in their workflow to make their narratives more robust rather than only post-hoc in evaluation.

That said, a useful distinction made here is to separate claims based on data/citations from claims that are inference or speculation. The latter of course are important for proposing parsimonious models that one can use as a theoretical framework for subsequent hypothesis generation or orthogonal testing. One awesome application of this in a future machine-readable world would be a forward-looking comparison of inference and speculation type claims with results from the rest of the corpus not just within-paper support.

Not at all a surprise is the increased coverage by LLMs compared to a few human reviewers who indeed likely only have expertise that covers a fraction of any interdisciplinary or collaborative work (the expertise of many authors went into a paper - the expertise of a reviewer is unlikely to span those).

Another thing that strikes me as invaluable here is the implicit and uncited result pairs that really will be essentially to generate knowledge graphs that can more usefully be built upon by new science/other scientists and far less biased than citation graphs.

Finally, as someone who spent many years invested in making the flip to the new eLife model (eliminating accept/reject editorial decisions), I was particularly interested in figure 2a comparing the old and new models. Even if unsupported claims had been dramatically higher or even high in an absolute sense, it would highlight the benefit of public reviews for such content rather than the same content being rejected and published as-is with a stamp of editorial acceptance elsewhere. However it's striking that even with many more claims evaluated by OpenEval, the unsupported claims are low whether authors decide when to stop revising their papers or when acceptance is gated by editors.

I think I am understanding the reason behind why a substance can only be gas or solid at low temperatures but I wanted to confirm with you. Due to the low temperatures they cant move or interact as normal so their particles are reliant on the pressure which is either pulling them apart with a vacuum so drastically that they become a gas or putting them under so much pressure that they get forced together to take up ass little room as possible.

To an extent, this makes me think back to the problem highlighted by Louca and Pennell (2020) of there being, for any given phylogeny of extant species only, large "congruence classes" of disparate diversification scenarios that could produce the observed trees. Could it be that this is still a problem that is faced by neural networks, as well as the more classical likelihood methods? Perhaps GNNs and the architectures you outline here could excel at inferring more localized (e.g. branch-specific, or tree-heterogeneous) diversification dynamics, rather than estimating tree-wide parameters under a global model?

eLife Assessment

This fundamentally significant study provides the first genome-wide characterization of H3K115 acetylation and identifies a striking and previously unappreciated association of this globular-domain histone modification with fragile nucleosomes at CpG island promoters, active enhancers, and CTCF binding sites. While the work is largely descriptive and correlative in nature the evidence is compelling. The authors present multiple, orthogonal genomic and biochemical analyses that consistently support their central conclusions.

Reviewer #1 (Public review):

Summary

The authors set out to define the genomic distribution and potential functional associations of acetylation of histone H3 lysine 115 (H3K115ac), a poorly characterized modification located in the globular domain of histone H3. Using native ChIP-seq and complementary genomic approaches in mouse embryonic stem cells and during differentiation to neural progenitor cells, they report that H3K115ac is enriched at CpG island promoters, active enhancers, and CTCF binding sites, where it preferentially localizes to regions containing fragile or subnucleosomal particles. These observations suggest that H3K115ac marks destabilized nucleosomes at key regulatory elements and may serve as an informative indicator of chromatin accessibility and regulatory activity.

Strengths

A major strength of this study is its focus on a histone post-translational modification in the globular domain, an area that has received far less attention than histone tail modifications despite strong evidence from structural and in vitro studies that such marks can directly influence nucleosome stability. The authors employ a wide range of complementary genomic analyses-including paired-end ChIP-seq, fragment size-resolved analyses, contour (V-) plots, and sucrose gradient fractionation-to consistently support the association of H3K115ac with fragile nucleosomes across promoters, enhancers, and architectural elements. The revised manuscript is careful in its interpretation and provides a coherent and internally consistent picture of how H3K115ac differs from other acetylation marks such as H3K27ac and H3K122ac. The datasets generated will be valuable to the chromatin community as a resource for further exploration of nucleosome dynamics at regulatory elements.

Weaknesses

The conclusions are largely correlative. While the authors provide strong evidence for the localization of H3K115ac to fragile nucleosomes, the work does not directly test whether this modification causally contributes to nucleosome destabilization or regulatory function in vivo. Questions regarding the enzymes responsible for depositing or removing H3K115ac and its direct functional consequences therefore remain open.

Overall assessment and impact

Overall, the authors largely achieve their stated aims by providing a detailed and well-supported characterization of H3K115ac distribution in mammalian chromatin and its association with fragile nucleosomes at regulatory elements. While mechanistic insight remains to be established, the study introduces a compelling new perspective on globular-domain histone acetylation and highlights H3K115ac as a potentially useful marker for identifying functionally important regulatory regions. The work is likely to stimulate further mechanistic studies and will be of broad interest to researchers studying chromatin structure, transcriptional regulation, and genome organization.

Reviewer #2 (Public review):

Summary:

Kumar et al. aimed to assess the role of the understudied H3K115 acetylation mark, which is located in the nucleosomal core. To this end, the authors performed ChIP-seq experiments of H3K115ac in mouse embryonic stem cells as well as during differentiation into neuronal progenitor cells. Subsequent bioinformatic analyses revealed an association of H3K115ac with fragile nucleosomes at CpG island promoters, as well as with enhancers and CTCF binding sites. This is an interesting study, which provides important novel insights into the potential function of H3K115ac. However, the study is mainly descriptive, and functional experiments are missing.

Strengths:

(1) The authors present the first genome-wide profiling of H3K115ac and link this poorly characterized modification to fragile nucleosomes, CpG island promoters, enhancers, and CTCF binding sites.

(2) The study provides a valuable descriptive resource and raises intriguing hypotheses about the role of H3K115ac in chromatin regulation.

(3) The breadth of the bioinformatic analyses adds to the value of the dataset

Comments on revisions:

The authors sufficiently addressed my concerns.

Reviewer #3 (Public review):

Summary:

Kumar et al. examine the H3K115 epigenetic mark located on the lateral surface of the histone core domain and present evidence that it may serve as a marker enriched at transcription start sites (TSSs) of active CpG island promoters and at polycomb-repressed promoters. They also note enrichment of the H3K115ac mark is found on fragile nucleosomes within nucleosome-depleted regions, on active enhancers and CTCF bound sites. They propose that these observations suggest that H3K115ac contributes to nucleosome destabilization and so may servers a marker of functionally important regulatory elements in mammalian genomes.

Strengths:

The authors present novel observations suggesting that acetylation of a histone residue in a core (versus on a histone tail) domain may serve a functional role in promoting transcription in CPG islands and polycomb-repressed promoters. They present a solid amount of confirmatory in silico data using appropriate methodology that supports the idea that H3K115ac mark may function to destabilize nucleosomes and contribute to regulating ESC differentiation. These findings are quite novel.

Weaknesses:

Additional experiments to confirm specificity of the antibodies used have been done, improving confidence in the study.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public reviews):

(1) The absence of replicate paired-end datasets limits confidence in peak localization.

The reviewer was under the impression that that we did not perform biological replicates of our ChIP-seq experiments. All ChIP-seq (and ATAC-seq) experiments were performed with biological replicates and the Pearson’s correlations (all >0.9) between replicates were provided in Supplementary Table 1. We had indicated this in the text and methods but will try to make this even clearer.

(2) The analyses are primarily correlative, making it difficult to fully assess robustness or to support strong mechanistic conclusions.

Histone modifications are difficult to alter genetically because of the high copy number of histone genes and inhibition of HATs/HDACs in general leads to alterations in other histone modifications. It is an inherent challenge in establishing causality of histone modifications, especially histone acetylation marks.

(3) Some claims (e.g., specificity for CpG islands, "dynamic" regulation during differentiation) are not fully supported by the analyses as presented.

We have modified the text in response to this point. The new text reads: “Non-CGI promoters have lower overall levels of transcription compared to CGI promoters, and for this promoter class H3K115ac enrichment detected by ChIP is only really seen for the highest quartile of transcription (4SU) quartile of expression (Figure 1G). CGI promoters on the other hand, exhibit significant levels of detected H3K115ac even for the lowest quartile of expression. These results suggest a special link between CGI promoters and H3K115ac”.

(4) Overall, the study introduces an intriguing new angle on globular PTMs, but additional rigor and mechanistic evidence are needed to substantiate the conclusions.

We agree that the paper does not provide mechanistic details or solid causality of H3K115ac. We have only emphasized the potential role of H3K115ac in nucleosome fragility based on our in vivo data and previously published in-vitro experiments (Manohar et.al., 2009, Chatterjee et. al., 2015). We do provide the evidence that H3K115ac is enriched on subnucleosomal particles via sucrose gradient sedimentation of MNase-digested chromatin (Figure 3C-D).

Reviewer #2 (Public review):

(1) I am not fully convinced about the specificity of the antibody. Although the experiment in Figure S1A shows a specific binding to H3K115ac-modified peptides compared to unmodified peptides, the authors do not show any experiment that shows that the antibody does not bind to unrelated proteins. Thus, a Western of a nuclear extract or the chromatin fraction would be critical to show. Also, peptide competition using the H3K115ac peptide to block the antibody may be good to further support the specificity of the antibody. Also, I don't understand the experiment in Figure S1B. What does it tell us when the H3K115ac histone mark itself is missing? The KLF4 promoter does not appear to be a suitable positive control, given that hundreds of proteins/histone modifications are likely present at this region. It is important to clearly demonstrate that the antibody exclusively recognizes H3K115ac, given that the conclusion of the manuscript strongly depends on the reliability of the obtained ChIP-Seq data.

ChIP-qPCR in S1B includes competition from native chromatin and shows high specificity to its target. We have provided antibody validation in three ways:

- Western blot with dot-blot of synthetic peptides (Figure S1A).

- Western blots with Whole cell extracts (Figure 4D).

- ChIP-qPCR on native chromatin spiked with a cocktail of synthetic mono-nucleosomes, each carrying a single acetylation and a specific barcode (SNAP-ChIP K-AcylStat Panel).

We could not include H3K115ac marked nucleosomes as they are not available in the panel. Figure S1B shows that the H3K115ac antibody exhibits negligible binding to known K-acyl marks, comparable to an unmodified nucleosome. Because of the absence of a H3K115ac modified barcoded nucleosome, we used the KLF4 promoter from mESCs as a positive control, in agreement with ChIP-seq signal shown in the genome browser profile (Figure 1E), the KLF4 promoter shows a significantly higher signal than the gene body.

(2) The association of H3K115ac with fragile nucleosomes is based on MNase-sensitivity and fragment length, which are indirect methods and can have technical bias. Experiments that support that the H3K115ac modified nucleosomes are indeed more fragile are missing.

We have performed ChIP-seq on MNase digested mESC chromatin fractionated on sucrose gradients and this shows that H3K115ac is enriched in fractions containing sub-nucleosomal and fragile nucleosomes but depleted in fractions containing stable nucleosomes (Figure 3D).

(3) The comparison of H3K115ac with H3K122ac and H3K64ac relies on publicly available datasets. Since the authors argue that these marks are distinct, data generated under identical experimental conditions would be more convincing. At a minimum, the limitations of using external datasets should be discussed.

H3K64ac and H3K122ac datasets were generated by us in a previous publication (Pradeepa et. al., 2016) using same native MNase ChIP protocol as used here. The ChIP-seq datasets for H3K122ac and H3K27ac are processed in an identical manner, with the same computational pipelines, to the H3K115ac data sets generated in this paper.

(4) The enrichment of H3K115ac at enhancers and CTCF binding sites is notable but remains descriptive. It would be interesting to clarify whether H3K115ac actively influences transcription factor/CTCF binding or is a downstream correlate.

We agree with the reviewer’s comment, but we have not claimed causality.

(5) No information is provided about how H3K115ac may be deposited/removed. Without this information, it is difficult to place this modification into established chromatin regulatory pathways.

Due to broad target specificity, redundancies and crosstalk among different classes of HATs and HDACs, it is not tractable to answer this question in the current manuscript.

Reviewer #3 (Public reviews):

Reviewer 3 is mistaken in thinking our ChIP experiments are performed under cross-linked conditions. As clearly stated in the main text and methods, all our ChIP-seq for histone modifications is done on native MNase-digested chromatin – with no cross-linking. This includes the spike-in experiment shown in Fig S1B to test H3K115ac antibody specificity against the bar-coded SNAP-ChIP® K-AcylStat Panel from Epicypher. We could not include H3K115ac bar-coded nucleosomes in that experiment since they are not available in the panel.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) I have two primary concerns that resound through the entire paper:

(a) Overall, the manuscript is making strong claims based on entirely correlative datasets. No quantitative analyses are performed to demonstrate co-occupancy/localization. Please see more detailed descriptions below.

Our responses to specific points are provided against each comment below.

(b) Lack of paired-end replicates for H3K115ac ChIP-seq. While the reviewer token for the deposited data was not made accessible to me, looking at Supplementary Table 1, it appears there are two H3K115ac ChIP-seq datasets. One is paired-end and is single-read. So are peaks called with only one replicate of PE? Or are inaccurate peaks called with SR datasets? Either way, this is not a rigorous way to evaluate H3K115ac localization.

We are sorry that this reviewer was not able to access the data – the token for the GEO accession was provided for reviewers at the journal’s request. All ChIP-seq (and ATAC-seq) experiments (paired and single-end) were performed with two biological replicates and the Pearson’s correlations (all >0.9) between replicates were provided in Supplementary Table 1. This was indicated in both the main text and in the methods. In the revised manuscript we have tried to make this even clearer and have put the relevant Pearsons coefficient (r) into the text at the appropriate places. For the reviewer’s information, here is the complete list of data samples in the GEO Accession:

Author response Image 1.

While I agree that H3K115ac occupancy is high at +CGIs, the authors downplay that H3K122ac and H3K27ac is also more highly enriched at these locations (page 7, last sentence of first paragraph). I imagine this is all due to the more highly transcribed nature of these genes. Sub-stratifying the K27ac and K122ac by transcription (as in Figure 1G) would help to demonstrate a unique nature of H3K115ac. But even better would be to do an analysis that plots H3K115ac enrichment vs transcription for every individual gene rather than aggregate analyses that are biased by single locations. For example, make an XY scatterplot of RNAPII occupancy or 4SU-seq signal vs H3K115ac level, where each point represents a single gene. Because the interpretation that it is CGI-based and not transcription is confounded with the fact that -CGI are more lowly transcribed. So, looking at Figure 1G, even the -CGI occupancy of H3K115ac is correlated with transcription, but it is just more lowly transcribed.

We thank the reviewer for these suggestions but point out that Figure 1G shows H3K115ac signal for CGI+ and CGI– TSS that are matched for expressions levels (quartiles of 4SU-seq). Fig 1F shows that H3k115ac is much more of a discriminator between CGI+ and – than H3K27ac or H3K122ac.

(2) H3K115ac, H3K27ac, and H3K122ac are all more enriched (in aggregate) at +CGI locations (Fig 1F); so do these locations just have more positioned nucleosomes? More H3.3? So that these PTMs are just more enriched due to the opportunity?

Positioned nucleosomes are generally found downstream of the TSS of active CpG island promoters, so what the reviewer suggests may well account for the relative enrichment of H327ac and H3K122ac at CGI+ vs CGI- promoters in Fig.1F. But H3K115ac localisation is distinct, with the peak at the nucleosome-depleted region not the +1 nucleosome. This is also confirmed by the contour plots in Fig 3. Our observation is also not explained by an enrichment of H3.3 at CGI promoters, since we show that H3K115ac is not specific to H3.3 (Fig 4D).

(3) The authors note in paragraph 2 of page 7 that "H3K115ac does not scale linearly with gene expression..." but the authors never show a quantification of this; stratification in four clusters is not able to make a linear correlation. Furthermore, in the second line of page 7, the authors state that the levels do generally correlate with transcription. To claim it is a specific CGI link and not transcription is tricky, but I encourage the authors to consider more quantifiable ways, rather than correlations, to demonstrate this point, if it is observed.

We thank the reviewer for this comment, and taking it into consideration, we have decided to re-phrase this paragraph. The new text reads: “Non-CGI promoters have lower overall levels of transcription compared to CGI promoters, and for this promoter class H3K115ac enrichment detected by ChIP is only really seen for the highest quartile of transcription (4SU) quartile of expression (Figure 1G). CGI promoters on the other hand, exhibit significant levels of detected H3K115ac even for the lowest quartile of expression. These results suggest a special link between CGI promoters and H3K115ac”.

(4) The authors claim on page 7 that "on average, transcription increased from TSS that also gained H3K115ac but to a modest extent, compared with the more substantial loss of H3K115ac from downregulated TSS". However, both upregulated and downregulated are significant; the difference in magnitude could simply be due to more highly or more lowly transcribed locations, meaning that fold change could be more robustly detected. I caution the authors to substantiate claims like this rather than stating a correlation.

We thank the reviewer for this comment which relates to the data in Fig 2A. It is Fig. 2B shows that the association of H3K115ac loss with downregulation is statistically stronger than H3K115ac gain with upregulation, but only for CGI promoters. With regard to the text on the original pg 7 that is referred to, we have now reworded this to read “Average levels of transcription increased from TSS that also gained H3K115ac, and there was loss of H3K115ac from downregulated TSS (Figure 2A).”

(5) For Figure 2C, the authors argue that H3K115ac correlate with bivalent locations. So this is all qualitative and aggregate localization; please quantitatively demonstrate this claim.

Figure S2D provides statistics for this (observed/expected and Fishers exact test).

(6) The authors claim in Figure 2 that H3115ac is dynamic during differentiation (title of Figure 2). However, there are locations that gain and lose, or maintain H3K115ac. In fact, the most discussed locations are H3K115ac with no change (2C); which means it is NOT dynamic during differentiation. So what is the message for the role during differentiation? From Supplemental Table 1, it appears there is a single ChIP experiment for H3K115ac in NPC, and it is a single read. So this is also a difficult claim with one replicate. Related to this, in S2A, the authors show K115ac where there is no change in transcription; so what is the role of H3K115ac at TSSs relevant to differentiation - it is at both locations changed and unchanged in transcription, but H3K115ac levels itself do not change at these subsets. So, how is this dynamic? This is very confusing, and clearer analyses and descriptions are necessary to deconvolute these data.

We apologise for the misleading title for Figure 2. This has now been amended to “Changes in H3K115ac during differentiation”. The message of this figure is that whilst changes in H3K115ac at TSS are small (panels A-C), at enhancers the changes are much more dramatic (panel D). The reviewer is incorrect about the number of replicates for NPCs – there are two biological replicates (see response to point 1b).

(7) The authors go on to examine H3K115ac enrichment on fragile nucleosomes through sucrose gradient sedimentation. A control for H3K27ac or H3K122ac would be nice for comparison.

We do not have the material available to perform these experiments

(8) When discussing Figures 3 and SF3, the authors mention performing a different MNase for a second ChIP. Showing the MNase distribution for both the more highly digested and the lowly digested would be nice. a) Related to the above, the authors show input in SF3E to argue that the difference in H3K115ac vs H3K27ac is not due to the library, but they do not show the MNase digestion patterns, which is more important for this argument.

Input libraries (first two graphs of FigS3E) are the MNase-digested chromatin. Comparison of nucleotide frequencies from millions of reads is more robust method than the fragment length patterns.

(9) The authors move on to examine H3K115ac at enhancers. Just out of curiosity, given what was found at promoters, is H3K115ac enriched at +CGI enhancers? And what is the correlation with enhancer transcription?

This is an interesting point, but the number of enhancers associated with CGI is not very high and so we did not focus on this. We have not analysed a correlation with eRNAs in this paper.

(10) The authors state on page 14 that the most frequent changes in H3K115ac during differentiation are at these enhancers. So do these changes connect with differentiation-specific genes, and/or genes that have altered transcription during differentiation? Just trying to understand the functional role.

Given the challenges of connecting enhancers with target genes, we have not addressed this question quantitatively. However, we draw the reviewer’s attention to the Genome Browser shots in Figures 2D and S2C, which show clear gain of H3K115ac (and ATAC-seq peaks) at intra and intergenic regions close to genes whose transcription is activated during the differentiation to NPCs.

(11) Related, at the end of page 14, the authors state that the changes in H3K115ac correlate with changes in ATAC-seq; I imagine this dynamic is not unique for H3K115ac and this is observed for other PTMs (H3K27ac), so assessing and clarifying this, to again get to the specific interest of H3K115ac, would be ideal.

We have not claimed that chromatin accessibility is unique to H3K115ac. It is the location of H3K115ac which is found inside the ATAC-seq peak region while H3K27ac is found only upstream/downstream of the ATAC peak that is so striking. This is apparent in Fig 4C.

(12) The authors examine levels of H3K115ac in H3.3 KO cell lines via western blot (Figure 4D), but no replicates and/or quantification are shown.

We now provide a biological replicate for the Western Blot (new FigS4H) together with an image of the whole gel for the data in Fig 4D

(13) In Figure S4 and at the end of page 17, the authors are arguing that there is a link to pioneer TF complexes, based on Oct4 binding. First, while Oct4 has pioneering activity, not all Oct4 sites (or motifs) are pioneering; this has been established. So if you want to use Oct4, substratifying by pioneer vs no pioneer is necessary. Second, demonstrating this is unique to pioneer and not to non-pioneer TFs would be an important control.

In response to the reviewer’s comment, we have removed the term “pioneer” from the manuscript.

(14) Minor point: Figure 4 A and B, there are some formatting issues with the scale bars.

We thank the reviewer for pointing this out, and the errors have been corrected in the revised figure.

(15) Minor point is that it should be clear when single replicates of data are used and when PE/SR sequences are combined or which one is used in each analysis, as this was hard to discern when reading the paper and figure legends.

We have clearly stated in the text that, after Figure2, we repeated all experiments in paired-end mode. All processing steps are defined separately for single end and paired end datasets in the method section. Details of biological replicates are provided in Sup. Table 1. These concerns are also addressed in our response to Reviewer’s public comment-1.

(16) Minor point: it is surprising that different MNase and different units were used in the ChIP vs sucrose sedimentation. Could the authors clarify why?

Chromatin prep for sucrose gradients were done on a much larger scale than for ChIP-seq and required different setups to obtain the right level of MNase digestion.

(17) The authors note that fragile nucleosomes contain H2A.Z and H3.3, but they never perform an analysis of available data to demonstrate a correlation (or better a quantifiable correlation) between H3K115ac occupancy and these marks at the locations they identify H3K115ac.

Since have shown (Fig. 4) that depletion of H3.3 does not affect overall levels of H3K115ac, we do not think there is value in further quantitative correlative analyses of H3K115ac and variant histones.

(18) Minor point: What is the overlap in peaks for H3K115ac, H3K122ac, and H3K27ac (Figure 1C)?

Nearly all H3K115ac peaks overlap with H3K122ac and/or H3K27ac. Its most distinct properties are its association with CGI promoters, fragile nucleosomes and its unique localisation within the NDRs, three points that the manuscript is focussed on.

Reviewer #3 (Recommendations for the authors):

(1) The western blot results in Figure 4D probing for H3, H3.3, and H3K115ac use Ponceau S staining, presumably of an area of the membrane where histones might be expected to migrate, as a measure of loading. However, the Ponceau S bands appear uniformly weaker in the H3.3KO lanes, yet despite this, blotting with H3.3 antibody detects a band in H3.3 knockout ESCs, suggesting that the antibody does not have a high degree of specificity. Again, a blocking experiment with appropriate peptides would instill more confidence in the specificity of these reagents, and/or the authors could provide independent validation of the knockout model to differentiate between a partial knockout or antibody cross-reactivity (e.g., by Sanger sequencing).

In a revised Fig. S4H we now show the whole gel corresponding to this blot but including co-staining with an antibody for H4 to provide a better loading control. We also provide a biological replicate of this Western blot in the lower panel of Fig. S4H.

(2) The manuscript would benefit from in vitro follow-up and validation, but if the authors intend to keep the manuscript primarily in silico, I suggest dedicating a few lines in each section to explain the plots, their axes, and their purpose, as well as to assist with interpretation, rather than directly discussing the results. This would make the manuscript more accessible and understandable for a broader audience in the field of epigenetics.

In the revised version, we have tried to improve the text to make the data more accessible to a broad audience.

I'm noticing in multiple figures the annotations of domains and the labelling of silk protein types are pixelated and unreadable inside the figure. This is something to note when exporting the figures in the final paper. I would consider using simpler symbols to signify the different classes of spider silk proteins containing only the name of the protein family.

Similar to my comment about not referring to extant proteins as ancestral, I would not refer to species that are currently alive as "ancient". Early-diverging is more accurate.

I noticed multiple references to these newly discovered extant proteins in an early diverging spider lineage as "ancestral proteins". I would steer clear from that language. Ancestral proteins implies that these proteins are no longer extant, and that they precede the existence of modern proteins. These are usually lost to time, but the term is reserved for proteins created with ancestral sequence reconstruction or archeologically recovered DNA. I would instead refer to these proteins as early-diverging, or belonging to spiders from an early-diverging lineage of Araneae.

I noticed that red arrows are mentioned in the figure, but I am not seeing those arrows in the figure itself. It might work to simply highlight the name of the taxon in red instead.

eLife Assessment

Studying the biological roles of polyphosphates in metazoans has been a longstanding challenge to the field given that the polyP synthase has yet to be discovered in metazoans. This important study capitalizes on the sophisticated genetics available in the Drosophila system and uses a combination of methodologies to start to tease apart how polyphosphate participates in Drosophila development and in the clotting of Drosophila hemolymph. The data validating one of these tools (cyto-FLYX ) are solid and well-documented and they will open up a field of research into the functional roles of polyP in a metazoan model. The other tools for tissue specific knockdown of polyP (Mito-FLYX, ER-FLYX, and Nuc-FLYX) have not yet been validated but will be invaluable to the field when they are.

Reviewer #2 (Public review):

Summary:

The authors of this paper note that although polyphosphate (polyP) is found throughout biology, the biological roles of polyP have been under-explored, especially in multicellular organisms. The authors created transgenic Drosophila that expressed a yeast enzyme that degrades polyP, targeting the enzyme to different subcellular compartments (cytosol, mitochondria, ER, and nucleus, terming these altered flies Cyto-FLYX, Mito-FLYX, etc.). The authors show the localization of polyP in various wild-type fruit fly cell types and demonstrate that the targeting vectors did indeed result in expression of the polyP degrading enzyme in the cells of the flies. They then go on to examine the effects of polyP depletion using just one of these targeting systems (the Cyto-FLYX). The primary findings from depletion of cytosolic polyP levels in these flies is that it accelerates eclosion and also appears to participate in hemolymph clotting. Perhaps surprisingly, the flies seemed otherwise healthy and appeared to have little other noticeable defects. The authors use transcriptomics to try to identify pathways altered by the cyto-FLYX construct degrading cytosolic polyP, and it seems likely that their findings in this regard will provide avenues for future investigation. And finally, although the authors found that eclosion is accelerated in pupae of Drosophila expressing the Cyto-FLYX construct, the reason why this happens remains unexplained.

Strengths:

The authors capitalize on the work of other investigators who had previously shown that expression of recombinant yeast exopolyphosphatase could be targeted to specific subcellular compartments to locally deplete polyP, and they also use a recombinant polyP binding protein (PPBD) developed by others to localize polyP. They combine this with the considerable power of Drosophila genetics to explore the roles of polyP by depleting it in specific compartments and cell types to tease out novel biological roles for polyP in a whole organism. This is a substantial advance.

Weaknesses:

Page 4 of Results (paragraph 1): I'm a bit concerned about the specificity of PPBD as a probe for polyP. The authors show that the fusion partner (GST) isn't responsible for the signal, but I don't think they directly demonstrate that PPBD is binding only to polyP. Could it also bind to other anionic substances? A useful control might be to digest the permeabilized cells and tissues with polyphosphatase prior to PPBD staining, and show that the staining is lost.

In the hemolymph clotting experiments, the authors collected 2 ul of hemolymph and then added 1 ul of their test substance (water or a polyP solution). They state that they added either 0.8 or 1.6 nmol polyP in these experiments (the description in the Results differs from that of the Methods). I calculate this will give a polyP concentration of 0.3 or 0.6 mM. This is an extraordinarily high polyP concentration, and is much in excess of the polyP concentrations used in most of the experiments testing the effects of polyP on clotting of mammalian plasma. Why did the authors choose this high polyP concentration? Did they try lower concentrations? It seems possible that too high a polyP concentration would actually have less clotting activity than the optimal polyP concentration.

In the revised version of the manuscript, the authors have productively responded to the previous criticisms. Their new data show stronger controls regarding the specificity of PPBD with regard to its interaction with polyP. The authors also have repeated their hemolymph clotting experiments with lower polyP concentrations, which are likely to be more physiological.

Reviewer #3 (Public review):

Summary:

Sarkar, Bhandari, Jaiswal and colleagues establish a suite of quantitative and genetic tools to use Drosophila melanogaster as a model metazoan organism to study polyphosphate (polyP) biology. By adapting biochemical approaches for use in D. melanogaster, they identify a window of increased polyP levels during development. Using genetic tools, they find that depleting polyP from the cytoplasm alters the timing of metamorphosis, accelerationg eclosion. By adapting subcellular imaging approaches for D. melanogaster, they observe polyP in the nucleolus of several cell types. They further demonstrate that polyP localizes to cytoplasmic puncta in hemocytes, and further that depleting polyP from the cytoplasm of hemocytes impairs hemolymph clotting. Together, these findings establish D. melanogaster as a tractable system for advancing our understanding of polyP in metazoans.

Strengths:

• The FLYX system, combining cell type and compartment-specific expression of ScPpx1, provides a powerful tool for the polyP community.

• The finding that cytoplasmic polyP levels change during development and affect the timing of metamorphosis is an exciting first step in understanding the role of polyP in metazoan development, and possible polyP-related diseases.

• Given the significant existing body of work implicating polyP in the human blood clotting cascade, this study provides compelling evidence that polyP has an ancient role in clotting in metazoans.

Limitations:

• While the authors demonstrate that HA-ScPpx1 protein localizes to the target organelles in the various FLYX constructs, the capacity of these constructs to deplete polyP from the different cellular compartments is not shown. This is an important control to both demonstrate that the GTS-PPBD labeling protocol works, and also to establish the efficacy of compartment-specific depletion. While not necessary to do for all the constructs, it would be helpful to do this for the cyto-FLYX and nuc-FLYX.

• The cell biological data in this study clearly indicates that polyP is enriched in the nucleolus in multiple cell types, consistent with recent findings from other labs, and also that polyP affects gene expression during development. Given that the authors also generate the Nuc-FLYX construct to deplete polyP from the nucleus, it is surprising that they test how depleting cytoplasmic but not nuclear polyP affects development. However, providing these tools is a service to the community, and testing the phenotypic consequences of all the FLYX constructs may arguably be beyond the scope of this first study.

Editors' note: The authors have satisfactorily responded to our most major concerns related to the specificity of PPDB and the physiological levels of polyPs in the clotting experiments. We also recognise the limitations related to the depletion of polyP in other tissues and hope that these data will be made available soon.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Polymers of orthophosphate of varying lengths are abundant in prokaryotes and some eukaryotes, where they regulate many cellular functions. Though they exist in metazoans, few tools exist to study their function. This study documents the development of tools to extract, measure, and deplete inorganic polyphosphates in *Drosophila*. Using these tools, the authors show:

(1) That polyP levels are negligible in embryos and larvae of all stages while they are feeding. They remain high in pupae but their levels drop in adults.

(2) That many cells in tissues such as the salivary glands, oocytes, haemocytes, imaginal discs, optic lobe, muscle, and crop, have polyP that is either cytoplasmic or nuclear (within the nucleolus).

(3) That polyP is necessary in plasmatocytes for blood clotting in Drosophila.

(4) That ployP controls the timing of eclosion.

The tools developed in the study are innovative, well-designed, tested, and well-documented. I enjoyed reading about them and I appreciate that the authors have gone looking for the functional role of polyP in flies, which hasn't been demonstrated before. The documentation of polyP in cells is convincing as its role in plasmatocytes in clotting.

We sincerely thank the reviewer for their encouraging assessment and for recognizing both the innovation of the FLYX toolkit and the functional insights it enables. Their remarks underscore the importance of establishing Drosophila as a tractable model for polyP biology, and we are grateful for their constructive feedback, which further strengthened the manuscript.

Its control of eclosion timing, however, could result from non-specific effects of expressing an exogenous protein in all cells of an animal.

We now explicitly state this limitation in the revised manuscript (p.16, l.347–349). The issue is that no catalytic-dead ScPpX1 is available as a control in the field. We plan to generate such mutants through systematic structural and functional studies and will update the FLYX toolkit once they are developed and validated. Importantly, the accelerated eclosion phenotype is reproducible and correlates with endogenous polyP dynamics.

The RNAseq experiments and their associated analyses on polyP-depleted animals and controls have not been discussed in sufficient detail.  In its current form, the data look to be extremely variable between replicates and I'm therefore unsure of how the differentially regulated genes were identified.

We thank the reviewer for pointing out the lack of clarity. We have expanded our RNAseq analysis in the revised manuscript (p.20, l.430–434). Because of inter-sample variation (PC2 = 19.10%, Fig. S7B), we employed Gene Set Enrichment Analysis (GSEA) rather than strict DEG cutoffs. This method is widely used when the goal is to capture pathway-level changes under variability (1). We now also highlight this limitation explicitly (p.20, l.430–432) and provide an additional table with gene-specific fold change (See Supplementary Table for RNA Sequencing Sheet 1). Please note that we have moved RNAseq data to Supplementary Fig. 7 and 8 as suggested in the review.

It is interesting that no kinases and phosphatases have been identified in flies. Is it possible that flies are utilising the polyP from their gut microbiota? It would be interesting to see if these signatures go away in axenic animals.

This is an interesting possibility. Several observations argue that polyP is synthesized by fly tissues: (i) polyP levels remain very low during feeding stages but build up in wandering third instar larvae after feeding ceases; (ii) PPBD staining is absent from the gut except the crop (Fig. S3O–P); (ii) In C. elegans, intestinal polyP was unaffected when worms were fed polyP-deficient bacteria (2); (iv) depletion of polyP from plasmatocytes alone impairs hemolymph clotting, which would not be expected if gut-derived polyP were the major source and may have contributed to polyP in hemolymph. Nevertheless, we agree that microbiota-derived polyP may contribute, and we plan systematic testing in axenic flies in future work.

Reviewer #2 (Public review):

Summary:

The authors of this paper note that although polyphosphate (polyP) is found throughout biology, the biological roles of polyP have been under-explored, especially in multicellular organisms. The authors created transgenic Drosophila that expressed a yeast enzyme that degrades polyP, targeting the enzyme to different subcellular compartments (cytosol, mitochondria, ER, and nucleus, terming these altered flies Cyto-FLYX, Mito-FLYX, etc.). The authors show the localization of polyP in various wild-type fruit fly cell types and demonstrate that the targeting vectors did indeed result in the expression of the polyP degrading enzyme in the cells of the flies. They then go on to examine the effects of polyP depletion using just one of these targeting systems (the Cyto-FLYX). The primary findings from the depletion of cytosolic polyP levels in these flies are that it accelerates eclosion and also appears to participate in hemolymph clotting. Perhaps surprisingly, the flies seemed otherwise healthy and appeared to have little other noticeable defects. The authors use transcriptomics to try to identify pathways altered by the cyto-FLYX construct degrading cytosolic polyP, and it seems likely that their findings in this regard will provide avenues for future investigation. And finally, although the authors found that eclosion is accelerated in the pupae of Drosophila expressing the Cyto-FLYX construct, the reason why this happens remains unexplained.

Strengths:

The authors capitalize on the work of other investigators who had previously shown that expression of recombinant yeast exopolyphosphatase could be targeted to specific subcellular compartments to locally deplete polyP, and they also use a recombinant polyP-binding protein (PPBD) developed by others to localize polyP. They combine this with the considerable power of Drosophila genetics to explore the roles of polyP by depleting it in specific compartments and cell types to tease out novel biological roles for polyP in a whole organism. This is a substantial advance.

We are grateful to the reviewer for their thorough and thoughtful evaluation. Their balanced summary of our work, recognition of the strengths of our genetic tools, and constructive suggestions have been invaluable in clarifying our experiments and strengthening the conclusions.

Weaknesses:

Page 4 of the Results (paragraph 1): I'm a bit concerned about the specificity of PPBD as a probe for polyP. The authors show that the fusion partner (GST) isn't responsible for the signal, but I don't think they directly demonstrate that PPBD is binding only to polyP. Could it also bind to other anionic substances? A useful control might be to digest the permeabilized cells and tissues with polyphosphatase prior to PPBD staining and show that the staining is lost.

To address this concern, we have done two sets of experiments:

(1) We generated a PPBD mutant (GST-PPBD^Mut). We establish that GST-PPBD binds to polyP-2X FITC, whereas GST-PPBD^Mut and GST do not bind polyP₁₀₀-2X FITC using Microscale Thermophoresis. We found that, unlike the punctate staining pattern of GST-PPBD (wild-type), GST-PPBD^Mut does not stain hemocytes. This data has been added to the revised manuscript (Fig. 2B-D, p.8, l.151–165).

(2) A study in C.elegans by Quarles et.al has performed a similar experiment, suggested by the reviewer. In that study, treating permeabilized tissues with polyphosphatase prior to PPBD staining resulted in a decrease of PPBD-GFP signal from the tissues (2). We also performed the same experiment where we subjected hemocytes to GST-PPBD staining with prior incubation of fixed and permeabilised hemocytes with ScPpX1 and heat-inactivated ScPpX1 protein. We find that both staining intensity and the number of punctae are higher in hemocytes left untreated and in those treated with heat-inactivated ScPpX1. The hemocytes pre-treated with ScPpX1 showed reduced staining intensity and number of punctae. This data has been added to the revised manuscript (Fig. 2E-G, p.8, l.166-172).

Further, Saito et al. reported that PPBD binds to polyP in vitro, as well as in yeast and mammalian cells, with a high affinity of ~45µM for longer polyP chains (35 mer and above) (3). They also show that the affinity of PPBD with RNA and DNA is very low. Furthermore, PPBD could detect differences in polyP labeling in yeasts grown under different physiological conditions that alter polyP levels (3). Taken together, published work and our results suggest that PPBD specifically labels polyP.

In the hemolymph clotting experiments, the authors collected 2 ul of hemolymph and then added 1 ul of their test substance (water or a polyP solution). They state that they added either 0.8 or 1.6 nmol polyP in these experiments (the description in the Results differs from that of the Methods). I calculate this will give a polyP concentration of 0.3 or 0.6 mM. This is an extraordinarily high polyP concentration and is much in excess of the polyP concentrations used in most of the experiments testing the effects of polyP on clotting of mammalian plasma. Why did the authors choose this high polyP concentration? Did they try lower concentrations? It seems possible that too high a polyP concentration would actually have less clotting activity than the optimal polyP concentration.

We repeated the assays using 125 µM polyP, consistent with concentrations employed in mammalian plasma studies (4,5). Even at this lower, physiologically relevant concentration, polyP significantly enhanced clot fibre formation (Included as Fig. S5F–I, p.12, l.241–243). This reconfirms the conclusion that polyP promotes hemolymph clotting.

Author response image 1.

Reviewer #3 (Public review):

Summary:

Sarkar, Bhandari, Jaiswal, and colleagues establish a suite of quantitative and genetic tools to use Drosophila melanogaster as a model metazoan organism to study polyphosphate (polyP) biology. By adapting biochemical approaches for use in D. melanogaster, they identify a window of increased polyP levels during development. Using genetic tools, they find that depleting polyP from the cytoplasm alters the timing of metamorphosis, accelerating eclosion. By adapting subcellular imaging approaches for D. melanogaster, they observe polyP in the nucleolus of several cell types. They further demonstrate that polyP localizes to cytoplasmic puncta in hemocytes, and further that depleting polyP from the cytoplasm of hemocytes impairs hemolymph clotting. Together, these findings establish D. melanogaster as a tractable system for advancing our understanding of polyP in metazoans.

Strengths:

(1) The FLYX system, combining cell type and compartment-specific expression of ScPpx1, provides a powerful tool for the polyP community.

(2) The finding that cytoplasmic polyP levels change during development and affect the timing of metamorphosis is an exciting first step in understanding the role of polyP in metazoan development, and possible polyP-related diseases.

(3) Given the significant existing body of work implicating polyP in the human blood clotting cascade, this study provides compelling evidence that polyP has an ancient role in clotting in metazoans.

We sincerely thank the reviewer for their generous and insightful comments. Their recognition of both the technical strengths of the FLYX system and the broader biological implications reinforces our confidence that this work will serve as a useful foundation for the community.

Limitations:

(1) While the authors demonstrate that HA-ScPpx1 protein localizes to the target organelles in the various FLYX constructs, the capacity of these constructs to deplete polyP from the different cellular compartments is not shown. This is an important control to both demonstrate that the GTS-PPBD labeling protocol works, and also to establish the efficacy of compartment-specific depletion. While not necessary to do this for all the constructs, it would be helpful to do this for the cyto-FLYX and nuc-FLYX.

We confirmed polyP depletion in Cyto-FLYX using the malachite green assay (Fig. 3D, p.10, l.212–214). The efficacy of ScPpX1 has also been earlier demonstrated in mammalian mitochondria (6). Our preliminary data from Mito-ScPpX1 expressed ubiquitously with Tubulin-Gal4 showed a reduction in polyP levels when estimated from whole flies (See Author response image 2 below, ongoing investigation). In an independent study focusing on mitochondrial polyP depletion, we are characterizing these lines in detail  and plan to check the amount of polyP contributed to the cellular pool by mitochondria using subcellular fractionation. Direct phenotypic and polyP depletion analyses of Nuc-FLYX and ER-FLYX are also being carried out, but are in preliminary stages. That there is a difference in levels of polyP in various tissues and that we get a very little subscellular fraction for polyP analysis have been a few challenging issues. This analysis requires detailed, independent, and careful analysis, and thus, we refrain from adding this data to the current manuscript.

Author response image 2.

Regarding the specificity, Saito et.al. reported that PPBD binds to polyP in vitro, as well as in yeast and mammalian cells with a high affinity of ~45µM for longer polyP chains (35 mer and above) (3). They also show that the affinity of PPBD with RNA and DNA is very low. Further, PPBD could reveal differences in polyP labeling with yeasts grown in different physiological conditions that can alter polyP levels. Now in the manuscript, we included following data to show specificity of PPBD:

To address this concern we have done two sets of experiments:

We generated a PPBD mutant (GST-PPBD^Mut). Using Microscale Thermophoresis, we establish that GST-PPBD binds to polyP₁₀₀-2X-FITC, whereas, GST-PPBD^Mut and GST do not bind polyP₁₀₀-2X-FITC at all. We found that unlike the punctate staining pattern of GST-PPBD (wild-type), GST-PPBD^Mut does not stain hemocytes. This data has been added to the revised manuscript (Fig. 2B-D, p.8, l.151–165).

A study in C.elegans by Quarles et.al has performed a similar experiment suggested by the reviewer. In that study, treating permeabilized tissues with polyphosphatase prior to PPBD staining resulted in decrease of PPBD-GFP signal from the tissues (2). We also performed the same experiment where we subjected hemocytes to GST-PPBD staining with prior incubation of fixed and permeabilised hemocytes with ScPpX1 and heat inactivated ScPpX1 protein. We find that both intensity of staining and number of punctae are higher in hemocytes that were left untreated and the one where heat inactivated ScPpX1 was added. The hemocytes pre-treated with ScPpX1 showed reduced staining intensity and number of punctae. This data has been added to the revised manuscript (Fig. 2E-G, p.8, l.166-172).

(2) The cell biological data in this study clearly indicates that polyP is enriched in the nucleolus in multiple cell types, consistent with recent findings from other labs, and also that polyP affects gene expression during development. Given that the authors also generate the Nuc-FLYX construct to deplete polyP from the nucleus, it is surprising that they test how depleting cytoplasmic but not nuclear polyP affects development. However, providing these tools is a service to the community, and testing the phenotypic consequences of all the FLYX constructs may arguably be beyond the scope of this first study.

We agree this is an important avenue. In this first study, we focused on establishing the toolkit and reporting phenotypes with Cyto-FLYX. We are systematically assaying phenotypes from all FLYX constructs, including Nuc-FLYX, in ongoing studies

Recommendations for the authors:

Reviewing Editor Comment:

The reviewers appreciated the general quality of the rigour and work presented in this manuscript. We also had a few recommendations for the authors. These are listed here and the details related to them can be found in the individual reviews below.

(1) We suggest including an appropriate control to show that PPBD binds polyP specifically.

We have updated the response section as follows:

(a) Highlighted previous literature that showed the specificity of PPBD.

(b) We show that the punctate staining observed by PPBD is not demonstrated by the mutant PPBD (PPBD^Mut) in which amino acids that are responsible for polyP binding are mutated.

(c) We show that PPBD^Mut does not bind to polyP using Microscale Thermophoresis.

(d) We show that treatment of fixed and permeabilised hemocytes with ScPpX1 reduces the PPBD staining intensity and number of punctae, as compared to tissues left untreated or treated with heat-inactivated ScPpX1.

We have included these in our updated revised manuscript (Fig. 2B-G, p.8, l.151–157)

(2) The high concentration of PolyP in the clotting assay might be impeding clotting. The authors may want to consider lowering this in their assays.

We have addressed this concern in our revised manuscript. We have performed the clotting assays with lower polyP concentrations (concentrations previously used in clotting experiments with human blood and polyP). Data is included in Fig. S5F–I, p.12, l.241–243.

(3) The RNAseq study: can the authors please describe this better and possibly mine it for the regulation of genes that affect eclosion?

In our revised manuscript, we have included a broader discussion about the RNAseq analysis done in the article in both the ‘results’ and the ‘discussion’ sections, where we have rewritten the narrative from the perspective of accelerated eclosion. (p.15 l.310-335, p. 20, l.431-446).

(4) Have the authors considered the possibility that the gut microbiota might be contributing to some of their measurements and assays? It would be good to address this upfront - either experimentally, in the discussion, or (ideally) both.

This is an exciting possibility. Several observations argue that fly tissues synthesize polyP: (i) polyP levels remain very low during feeding stages but build up in wandering third instar larvae after feeding ceases; (ii) PPBD staining is absent from the gut except the crop (Fig. S3O–P); (iii) in C. elegans, intestinal polyP was unaffected when worms were fed polyP-deficient bacteria (2); (iv) depletion of polyP from plasmatocytes alone impairs hemolymph clotting, which would not be expected if gut-derived polyP were the major source and may have contributed to polyP in hemolymph. Nevertheless, microbiota-derived polyP may contribute, and we plan systematic testing in axenic flies in future work.

Reviewer #1 (Recommendations for the authors):

(1) While the authors have shown that the depletion tool results in a general reduction of polyP levels in Figure 3D, it would have been nice to show this via IHC. Particularly since the depletion depends on the strength of the Gal4, it is possible that the phenotypes are being under-estimated because the depletions are weak.

We agree that different Gal4 lines have different strengths and will therefore affect polyP levels and the strength of the phenotype differently.

We performed PPBD staining on hemocytes expressing ScPPX; however, we observed very intense, uniform staining throughout the cells, which was unexpected. It seems like PPBD is recognizing overexpressed ScPpX1. Indeed, in an unpublished study by Manisha Mallick (Bhandari lab), it was found that His-ScPpX1 specifically interacts with GST-PPBD in a protein interaction assay (See Author response image 3). Due to these issues, we refrained from IHC/PPBD-based validation.

Author response image 3.

(2) The subcellular tools for depletion are neat! I wonder why the authors didn't test them. For example in the salivary gland for nuclear depletion?

We have addressed this question in the reviewer responses. We are systematically assaying phenotypes from all FLYX constructs, including Mito-FLYX, and Nuc-FLYX, in ongoing independent investigations. As discussed in #1, a possible interaction of ScPpX and PPBD is making this test a bit more challenging, and hence, they each require a detailed investigation.

(a) Does the absence of clotting defects using Lz-gal4 suggest that PolyP is more crucial in the plasmatocytoes and for the initial clotting process? And that it is dispensible/less important in the crystal cells and for the later clotting process. Or is it that the crystal cells just don't have as much polyP? The image (2E-H) certainly looks like it.

In hemolymph, the primary clot formation is a result of the clotting factors secreted from the fat bodies and the plasmatocytes. The crystal cells are responsible for the release of factors aiding in successfully hardening the soft clot initially formed. Reports suggest that clotting and melanization of the clot are independent of each other (7). Since Crystal cells do not contribute to clot fibre formation, the absence of clotting defects using LzGAL4-CytoFLYX is not surprising. Alternatively, PolyP may be secreted from all hemocytes and contribute to clotting; however, the crystal cells make up only 5% hemocytes, and hence polyP depletion in those cells may have a negligible effect on blood clotting.

Crystal cells do show PPBD staining. Whether polyP is significantly lower in levels in the crystal cells as compared to the plasmatocytes needs more systematic investigation. Image (2E-H) is a representative image of the presence of polyP in crystal cells and can not be considered to compare polyP levels in the crystal cells vs Plasmatocytes.

(b) The RNAseq analyses and data could be better presented. If the data are indeed variable and the differentially expressed genes of low confidence, I might remove that data entirely. I don't think it'll take away from the rest of the work.

We understand this concern and, therefore, in the revised manuscript, we have included a broader discussion about the RNAseq analysis done in the article in both the ‘results’ and the ‘discussion’ sections, where we have rewritten the narrative from the perspective of accelerated eclosion. (p.15 l.310-335, p. 20, l.431-446). We have also stated the limitations of such studies.

(c) I would re-phrase the first sentence of the results section.

We have re-phrased it in the revised manuscript.

Reviewer #2 (Recommendations for the authors):

(1) The authors created several different versions of the FLYX system that would be targeted to different subcellular compartments. They mostly report on the effects of cytosolic targeting, but some of the constructs targeted the polyphosphatase to mitochondria or the nucleus.

They report that the targeting worked, but I didn't see any results on the effects of those constructs on fly viability, development, etc.

There is a growing literature of investigators targeting polyphosphatase to mitochondria and showing how depleting mitochondrial polyP alters mitochondrial function. What was the effect of the Nuc-FLYX and Mito-FLYX constructs on the flies?

Also, the authors should probably cite the papers of others on the effects of depleting mitochondrial polyP in other eukaryotic cells in the context of discussing their findings in flies.

We have addressed this question in the reviewer responses. We did not see any obvious developmental or viability defects with any of the FLYX lines, and only after careful investigation did we come across the clotting defects in the CytoFLYX. We are currently systematically assaying phenotypes from all FLYX constructs, including Mito-FLYX and Nuc-FLYX, in independent ongoing investigations.

We have discussed the heterologous expression of mitochondrial polyphosphatase in mammalian cells to justify the need for developing Mito-FLYX (p. 10, l. 197-200). In the discussion section, we also discuss the presence and roles of polyP in the nucleus and how Nuc-FLYX can help study such phenomena (p. 19, l. 399-407).

(2) The authors should number the pages of their manuscript to make it easier for reviewers to refer to specific pages.

We have numbered our lines and pages in the revised manuscript.

(3) Abstract: the abbreviation, "polyP", is not defined in the abstract. The first word in the abstract is "polyphosphate", so it should be defined there.

We have corrected it in the revised version.

(4) The authors repeatedly use the phrase, "orange hot", to describe one of the colors in their micrographs, but I don't know how this differs from "orange".

‘OrangeHot’ is the name of the LUT used in the ImageJ analysis and hence referred to as the colour

(5) First page of the Introduction: the phrase, "feeding polyP to αβ expression Alzheimer's model of Caenorhabditis elegans" is awkward (it literally means feeding polyP to the model instead of the worms).

We have revised it. (p.3, l.55-57).

(6) Page 2 of the Introduction: The authors should cite this paper when they state that NUDT3 is a polyphosphatase: https://pubmed.ncbi.nlm.nih.gov/34788624/

We have cited the paper in the revised version of the manuscript. (p.4, l. 68-70)

(7) Page 2 of Results: The authors report the polyP content in the third instar larva (misspelled as "larval") to five significant digits ("419.30"). Their data do not support more than three significant digits, though.

We have corrected it in the revised manuscript.

(8) Page 3 of Results (paragraph 1): When discussing the polyP levels in various larval stages, the authors are extracting total polyP from the larvae. It seems that at least some of the polyP may come from gut microbes. This should probably be mentioned.

This is an interesting possibility. Several observations argue that polyP is synthesized by fly tissues: (i) polyP levels remain very low during feeding stages but build up in wandering third instar larvae after feeding ceases; (ii) PPBD staining is absent from the gut except the crop (Fig. S3O–P); (ii) In C. elegans, intestinal polyP was unaffected when worms were fed polyP-deficient bacteria (2); (iv) depletion of polyP from plasmatocytes alone impairs hemolymph clotting, which would not be expected if gut-derived polyP were the major source and may have contributed to polyP in hemolymph. We mention this limitation in the revised manuscript (p.19-20, l. 425-433).

(9) Page 3 of Results (paragraph 2): stating that the 4% paraformaldehyde works "best" is imprecise. What do the authors mean by "best"?

We have addressed this comment in the revised manuscript and corrected it as 4% paraformaldehyde being better among the three methods we used to fix tissues, which also included methanol and Bouin’s fixative  (p.8, l. 152-154).

(10) Page 4 of Results (paragraph 2, last line of the page): The scientific literature is vast, so one can never be sure that one knows of all the papers out there, even on a topic as relatively limited as polyP. Therefore, I would recommend qualifying the statement "...this is the first comprehensive tissue staining report...". It would be more accurate (and safer) to say something like, "to our knowledge, this is the first..." There is a similar statement with the word "first" on the next page regarding the FLYX library.

We have addressed this concern and corrected it accordingly in the revised version of the manuscript (p.9, l. 192-193)

Reviewer #3 (Recommendations for the authors):

(1) The authors should include in their discussion a comparison of cell biological observations using the polyP binding domain of E. coli Ppx (GST-PPBD) to fluorescently label polyP in cells and tissues with recent work using a similar approach in C. elegans (Quarles et al., PMID:39413779).

In the revised manuscript, we have cited the work of Quarles et al. and have added a comparison of observations (p.19,l.408-410). In the discussion, we have also focused on multiple other studies about how polyP presence in different subcellular compartments, like the nucleus, can be assayed and studied with the tools developed in this study.

(2) The gene expression studies of time-matched Cyto-FLYX vs WT larvae is very intriguing. Given the authors' findings that non-feeding third instar Cyto-FLYX larvae are developmentally ahead of WT larvae, can the observed trends be explained by known changes in gene expression that occur during eclosion? This is mentioned in the results section in the context of genes linked to neurons, but a broader discussion of which pathway changes observed can be explained by the developmental stage difference between the WT and FLYX larvae would be helpful in the discussion.

We have included a broader discussion about the RNAseq analysis done in the article in both the ‘results’ and the ‘discussion’ sections, where we have rewritten the narrative from the perspective of accelerated eclosion. (p.15 l.310-335, p. 20, l.431-446). We have also stated the limitations of such studies.

(3) The sentence describing NUDT3 is not referenced.

We have addressed this comment and have cited the paper of NUDT3 in the revised version of the manuscript.(p.4, l. 68-70)

(4) In the first sentence of the results section, the meaning/validity of the statement "The polyP levels have decreased as evolution progressed" is not clear. It might be more straightforward to give an estimate of the total pmoles polyP/mg protein difference between bacteria/yeast and metazoans.

In the revised manuscript, we have given an estimate of the polyP content across various species across evolution to uphold the statement that polyP levels have decreased as evolution progressed (p. 5, l. 87-91).

(5) The description of the malachite green assay in the results section describes it as "calorimetric" but this should read "colorimetric?"

We have corrected it in the revised manuscript.

References

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Trying to keep oneself safe from death will not work to prevent them being cast to Hell at any moment.

Even if there are no obvious ways for someone to die, including them being healthy, they can still die and be sucked into Hell at any time.

I find that this is an incredibly sad way of thinking.

Oh my god??

Very interesting paper! I like that the authors looked at this problem that has been traditionally difficult to study in this way and attempted to use the tools available to bring it into the realm of something study-able!

I think this is a really important point, and I like that you showed conservation of binding thermodynamics for PfBDP1 with RMM23 as my biggest question throughout the manuscript was how you ensure that protein function is preserved when introducing this many sequence changes. These constraints seem like a strong step, as well as that control, but maybe still an important thing to keep in mind. For example, even if the binding pocket geometry is structurally conserved, mutations outside the pocket could still influence binding (e.g., through altered dynamics, stability of the fold, or long-range effects on binding energetics).

This is really powerful as it's allowing you to study proteins that otherwise were not able to be studied in this way. But related to this idea that AF2 isn't capturing conformational ensembles, are you thinking about the effect that stabilizing the protein might have on your ability to study biologically relevant conformational dynamics of the protein?

It would be interesting to know what the pLDDT values are for the wild-type proteins as well if you ran them through your structure prediction tool, just to see if there is a significant change. Interesting thought that pLDDT alone doesn't distinguish between well-behaved proteins and non-well-behaved proteins!

Why did you select the most sequence-diverse candidates? I could be wrong, but if the goal is just to stabilize the proteins while maintaining their other biological functions wouldn't you want to minimize changes? Also curious how many candidates you considered per protein and how many met the criteria before you selected the candidates you moved forward with.

all the way

The relationship between what is measured, how it's measured, and evolutionary diversification is always important and interesting. It would be useful to know how the previous aggregation metrics covary with yours. Are they simply more and less nuanced measurements of the same trait? Measuring fundamentally different things? Are they differentially sensitive to certain strains over others? Do the previous metrics show any association with genetic/evolutionary factors? I wonder if this might help refine the true phenotypic locus of selection.

reflective of the time

madonna whore complex. i wonder how he views his mother?

Right. Okay.

Good.

imitate

look into?

the definition of who is elite radically changes the problematic potential.

populism def

Steering wheel- In life any things guide me however one big thing would be my religious beliefs and faith. Brakes- Within life the fear of failing and not meeting expectations can hold me back. Gas- My relationships and goals move me forward in life. Windsheild wipers- Goals help me plan out my success to see clearly. Rearview mirrors- I look back on my achievements the most to help me keep going. Seats- The help I receive from anyone makes me feel comfortable. Passengers- God and my relationships such as friends and family help to keep me company.

I like this! Our world runs based on what we pay attention to, and what is attention and a willingness to do something about what is noticed but care? If we don't care about something, only the sensational or head turning is revealed to us-- it seems like we lose the everyday

It's interesting that the definition (or the impression) of the innovation/repair dichotomy leaves little room for maintenance. It's almost like we never see a plateau, or little course corrections visibly indicated for a technology simply because they might not be interesting. We're constantly looking for new pieces of information: things to be proud of and things to solve, but we don't quite let ourselves stay in that intermediary stage without feeling stagnant or getting bored.

It's interesting as a commentary for people too-- when we feel broken or in need of repair, there is nothing you can see on the outside that would indicate it. We kind of act like these finished pictures, and there's no way to really see us in construction (at least, in the construction's entirety).

It's interesting how this thinking is so dichotomous-- we oscillate between both the pain and beauty of what's broken and feel torn about what to believe (or more specifically, what's more helpful to believe). You can protect your peace al you want, but is it worth it to not see problems in the world without solutions and without an inherently positive twist on them?

Promising and reassuring for the readers privacy, however not clear if other types of sharing like with subprocessors or legal requests are still allowed.

I still don't think I fully understand this, but it's a bit easier to think about after doing the Kinetics Discussion worksheet.

If possible, can someone explain this in other words?

This is also the same sort of passive aggressive tone as from before, since he is coupling negotiations from a position of power and basically drawing a hard line that he will not come back unless the Colonel Anderson can guarantee his daughters' safety. But he couples it with a sort of innocent question about schooling as well. It's surprisingly educated in its delivery, since everytime it takes something or delivers a devastating statement, it also balances it with either an outright compliment or a mundane question to dilute the previous blow. It as a whole seems to be establishing a sort of idea that, yes, he's willing to return, but he's actually not, as he's demanding many things that the Colonel can't provide. It's basically a soft rejection of his offer by making it look instead of as a personal decision off of just a refusal to work for him, but by disguising it as a logical decision based on just common working conditions and employment, such as differing wages and benefits. Again, it seems surprisingly well put together and educated, which likely is also intentionally done to undermine the Colonel's position.

I feel like this is almost an attempt at flattery, trying to get a good feeling for the Colonel Anderson's reaction. Seeing as it would be a more positive comment, since he's blatantly saying that he was proud to have been owned by a Colonel, but it also shows a level of passive aggressive education as well. This is because he's asking to hear about the wages, basically challenging Colonel Anderson to actually offer better than anybody else can, as he had said. It makes the situation interesting simply because It's a subservient tone that then shifts into this demanding and negotiating tone, since he's basically acting as if he is still a slave before moving on to discuss wages and other benefits.

This is a very sad picture. They had so many dead people and students that they had to go in groups, its sad that they didn't get their own ceremony and go in peace.

The description of this picture is very heartbreaking. The mother having to walk around carrying her dead child while looking for a place to lay her to rest is sad.

This image puts you in a peaceful mindset, the lanterns along with the pretty background shows signs of hope.

This image put you into the chaotic setting, the fire surrounding the buildings and the burns on the people shows the destruction caused.

Self explanatory

Important when writing introduction

Did not know this

Make the book happy and put /hb

Important