I would make this a main figure — it seems almost like a graphical abstract of the entire paper and shows readers at a glance how different programs they might be familiar with can all work harmoniously. It might also be good to show this high-level view of the system before showing the more nitty-gritty views in Figure 2

You might want to mention to readers that Airtable also enables no-code automations (and you can use Zapier for some more complicated but still no-code automation) since some labs lack even basic scripting ability. You describe "no-code implementation" early in the manuscript, so it seems important to clarify exactly what can be done without code and what strictly requires it.

Separately, you could also mention that Airtable can communicate with Slack (I see Telegram in Supp Fig 3, but I believe Slack is probably more common among scientists), Google Drive, etc.

You might want to define this like you did for "single source of truth" since you're using it in a bit of a unique way. How do you define an atom (aka the smallest unit) in this context?

I'd link to the example base again here too — looking at that is probably the easiest way for someone to quickly understand the concept! You've laid it out really nicely

How did you do this survey? This seems like a useful dataset that others might want to cite if you provide some more methodological info (e.g., Who are the respondents? Did you post this on social media? Do you know that each respondent is from a different lab or might this not really represent 233 distinct labs?)

This is a really cool preprint! We use Airtable to run the internal aspects of our publishing system at Arcadia Science and would love to see more scientists taking advantage. Your example base is perfect for helping readers understand and adapt what you've built.

I've added some thoughts that I had while reading, hopefully they're helpful.

make sure your sources are relevant to the topic and main idea

Even though the writing uses special terms, it’s still written in a fair and professional way. The authors don’t let their personal feelings show, and they keep the language serious and formal

Each type is different some are more reliable or detailed than others. Knowing the difference helps me pick the right sources for my research when I know I can get a more accurate answer and understanding

Database is pretty useful, it organizes the information and makes it easier to look up specific topics, authors, or dates, so I can find what I need for my research more efficiently.

It’s important you know based on sources what information has been gathered and the truth of that topic

These materials that take information from original sources and then explain or talk about it in more detail. They more so focus on the original content to help people better understand it.

Basically original materials or evidence that come straight from the people or events being studied.

Our fathers who believed in God, even their ancestors trusted God, which makes it a tradition that continues and originates from morality and religion, and this is what characterizes the foundations of unionist Protestantism in Ulster where precisely the Christian faith is intimately linked to the defense of British identity and civil liberties. To summarize, this sentence gives them a feeling of carrying a legacy and defending a cause much bigger than themselves.

The paper concludes that the problem isn't just the noise itself, but a failure of governance and lack of resources to enforce laws. This suggests that technical solutions alone won't work without addressing the underlying political and economic issues.

This is a shocking statistic. It directly links high nighttime noise levels to a major health problem (insomnia) for a huge majority of residents. This clearly shows the convergence between the measured high noise levels and the perceived negative health impacts. This would be a great quote for Project 2 or 3.

Even in residential areas, the average daytime noise level (55.42 dB) is higher than the WHO's recommended maximum (50 dB). This is a specific piece of evidence showing the problem isn't just in commercial or transport areas; it's affecting people where they live.

The authors used a mix of quantitative like sound-level meter in the field survey and qualitative like questionnaires, interviews methods. This is called a mixed-methods approach and makes their findings more robust because it combines objective data with subjective human experience.

Noise isn't just annoying; it has four distinct categories of negative health impacts. This is a great, concise list of the specific health risks I can reference later. The physiological effects like increased blood pressure are especially surprising.

This sentence identifies the main research gap the study is trying to fill. It's not just about noise, but about comparing measured noise with what people feel in medium-sized cities in the Global South. This is the core purpose of the article.

Goals help to guide you in the process.

"the links carrying traffic between the extremities of a network and its core"

I found this article very interesting. I wouldn't have thought that someone would dedicate their study to trolls, but I guess there is an expert on everything out there somewhere. I would like to hear more about the in person interactions with trolls because it said that she met and conversed with 30. I want to know how these conversations go, does she pretend that she is also really into trolling and then at the end tell them that she is investigating them and almost troll them herself. I would watch a show that was along the lines of that.

[g32] argues that ignoring rarely stops harassment; abusers escalate to force a reaction. The piece advocates platform-level moderation and removals, shifting responsibility from victims to systems. This directly reinforces §7.4’s critique of “don’t feed the trolls” and grounds the chapter’s recommendation in reported cases rather than abstract principle.

The Jenkem prank shows that trolls know exactly how to weaponize sensationalism and exploit weaknesses in journalistic culture to make a point， if journalists treated troll-generated stories with more skepticism instead of just chasing clicks, would trolls lose most of their power?

This is such an important topic because of how impactful it is on students and their mental health. I wonder how music teachers can correct without isolating or giving a negative impression?

I like how caring and ethics is being combined here because they should be when dealing with humans. Inclusion shouldn't be just policy but a moral responsibility. I do wonder what the "feminine" significance is in this title.

I wonder if you could use music as a way to help regulate emotions and calm anxiety so the student is still participating? Either through listening to a calming song or making a beat.

This changes how I think about how important communication is for students with disabilities. Instead of looking at these behaviours as "good" or "bad" we should instead be looking at the causes and what this could be potentially communicating.

One connection I see is from the “Amy and Drew” story from class, where flexibility and understanding made inclusion work.

I am happy to be learning about this because this is something that I have not considered before. Allowing students to help each other is very important but how you frame it is just as or more so important. "Buddy burnout" is also a new concept I am interested in learning more about.

Paraprofessionals are also such a huge resource when it comes to having students with disabilities in your class. They know the students very well because they spend so much time one on one with the students and usually have very effective strategies on managing behaviors.

I see this in action when I am student teaching this semester with my co-op in beginning band. We try to ask lots of questions to the students to demonstrate their knowledge, have them demonstrate their skills, and have them interact with technology when being assessed for learning.

I never considered this idea before this reading. Students are much more aware than we realize as adults and as educators. I think including them to the extent that is appropriate is a great tool and helps them understand their role and value.

I know some classroom teachers in the beginning of the school year have the students come up with class rules to follow as a group. This is a great way to get students involved in the expectations of behavior. It also gives them a voice for how they would like to build their classroom community.

I think the best way to deal with trolls is to ignore them. I know it says in the text that ignoring doesn't work because they just come back stronger and more hostile. Even if they do continue I think it is still the best option because it doesn't satisfy them, when they get no reaction at all to their trolling they are left unsatisfied. From here you can block them, report them, or restrict them in some way so that you don't have to keep hearing from them.

In §7.4, I argue that the "don't feed the trolls" rule shifts responsibility to the targets. On Discord, I help keep a balance, not focusing solely on doxing threats that have escalated; what finally worked was friction and compliance: maIembe mode during spikes, cooldowns for new members, removal of prohibitive thresholds, and robust reporting. I also interpret §7.3 as the ethical distinction between the “increase” of protest trolling (e.g., flooding K-pop apps) and the “reduction” of cruelty (RIP). It would be helpful if the chapter explicitly identified conformity and power dynamics as key dividing lines between ridicule, resistance, and contempt.

I agree with the idea that just “not feeding the trolls” doesn’t always work. Sometimes ignoring them gives them more space to keep spreading hate, especially when the target is already being attacked or harassed. I think the article makes a good point that it’s unfair to put all the responsibility on the person being targeted.

This was a letter sent by the FBI to Martin Luther King Jr. It was sent to him in 1964 and detailed alleged sexual indiscretions that were committed by King. The letter implied that King should kill himself because his shame about his crimes should be too great for him to live with.

In the initial stages you want to be nimble. Scalable and complicated architecture makes that difficult.

But you also need for burst patterns as explained in one of the sub-comments.

I think that websites should just be very strict and ban people who do not follow the guidelines. If the moderation on a website is strict, it makes it much harder for a trolling culture to develop and thrive. As long as there is a zero tolerance policy for bullying, trolling on a website should be kept to a minimum.

computers and cold war politics shaped eachother

The belief in total control of the internet and having surveillance systems

Early internet wasnt as utilized at the start as it is now

ignores the problems during this time and focuses on the fun story

The internet grew from closed/private and public influences. (Military and countercultural)

Hype vs reality, author is talking about the over exaggerating of the internets impact

How recent and signifigant newfound technlogy has been

This version is very different. I suppose the changes to the speech are for clarity as it is more or less the same. I wonder if her descriptions of the crowd are accurate though. Did Sojourner Truth really bring the house down?

so depending on the industry the AI is being deployed, the company would assume contractual liability if the model operates outside of the intended purpose of the company. This would make AI companies assume more liability in low risk scenarios such as automation for routine tasks or cloud computing, and assume less liability in high risk scenarios such as automation in hospitals. But overall, the terms would be contractual and tort would be ad hoc. Oh and the contracts would be made by AI.

for - youtube - neuroscience - How the brain remembers and imagines - Donna Rose Addis - memory and imagination have the same basis

summary - Donna Rose Addis is a pioneer in a field that connects past memories to future imagination - Her research has demonstrated that the same brain region, the Default Mode Network is responsible for simulations of past memories as well as future imagination - It is theorirized that episodic memory is reactivated and reorganized for creating future simulations

for - adjacency - depression - double for positive future thinking - even more amongst males

for - examples - autobiographical simulations invoking past episodic memories for future (goal-seeking) - counterfactuals - reimagining the past to see how we could have done better - anterior hippocampus supports imagination of - detailed, coherent and novel events and encoding the simulation so we can recognize when the opportunity arises in the future - creative cognition - populations with memory impairments also suffer difficulty with future imagination - depression results in loss of specificity of memories

for - definition - Constructive Episodic Simulation Hypothesis - episodic memory is a constructive process that reactivates and reintegrates distributed information across the brain - these same memories can be used in new combinations for novel imagination of the future

for - adjacency - memory - imagination critical papers - 2007 - some types of Alzheimer patient cannot imagine the future

Vanilla = standard/default system Customizations are added later to meet specific business needs.

This explains two different hypotheses about how moonlight affects nocturnal animal behavior. The Predation Risk hypothesis suggests that animals reduce activity during bright moonlight to avoid predators that can see them easily. The Visual Acuity hypothesis argues that species that depend on sight to find food or detect danger become more active when the moon is bright, while those relying on other senses become less active.

"Who is the third who walks always beside you?" correlates with Ernest Shackleton's account during his last expedition. After his trek across South Georgia, he wrote, "it seemed to me often that we were four, not three" (pg. 211). His companions confirmed this eeriness of some type of mysterious presence. Eliot uses Shackleton's testimony to inject a brief moment of the uncanny and the potentially divine into the poem's landscape. The setting described by Eliot in this scene is more vague and ambiguous than Shackleton's certainty. The poem counts only two figures, yet, sees a "gliding wrapt in a brown mantle, hooded" (line 364). This figure is unrecognisable, evident from line 365: "I do not know whether a man or a woman." It could be: Christ, appearing to the disciples on the road to Emmaus (Luke 24), where they do not recognize him; a hallucination born of exhaustion and spiritual despair; or the holy spirit, present but unrecognized. Later, Eliot reiterates the question, this time in a more intensified manner. He writes, "But who is that on the other side of you?" (line 366). Eliot is suggesting not one, but multiple mysterious presences. This resonates with the Indian legend from Marudanayagam, where three seers become aware of a fourth, the Lord Vishnu himself. The passage enacts a similar spiritual arithmetic: the human count is wrong; the divine is present but unseen, or unacknowledged, in the waste land. It is a moment where the material world seems to be haunted by a spiritual reality that the inhabitants can sense but not comprehend.

Eliot constantly mentions tarot cards and myths with themes of fertility and sterility. There is also a big contrast between water and fire. In a poem filled with so many symbols and elements indicating destruction and//or a form of darkness, water has served as a palette cleanser and a symbol of life, spirit, and purification. And so, it is interesting how here, there is "no water but only rock." Eliot later writes on lines 338-339, "If there were only water amongst the rock / Dead mountain mouth of carious teeth that cannot spit." This image perfectly encapsulates the sterility in this section and the true "waste land." It's also interesting how the title of this section is literally "What the Thunder Said," and yet, there is only "dry sterile thunder without rain" (line 342). In Themis, Harrison explains that thunder was not just a sound, but a manifestation of divine power, or mana, a force that brought a life-giving rain. In contrast, the thunder in this section is empty and a form of revelation without its substance. It promises meaning but delivers none. In this way, it mirrors a modern world where ancient symbols have lost their power. Finally, from Hermit Thrush, Eliot states that the hermit thrush was "the sweetest singer of all American birds." Its song is not one of mere prettiness, but of "tranquil clearness of tone and exalted serenity." By introducing it here, Eliot holds up an ideal of pure, natural, spiritual music, a stark contrast to the "dry grass singing" and the "cicada." The thrush's song is inseparable from the "sound of water over a rock." The "Drip drop drip drop drop drop drop" is both the sound of water and the rhythm of the bird's song. This fusion creates a single, transcendent symbol for grace, creativity, and spiritual renewal.

The idea of a river sweating is peculiar. Sweating is the release of a liquid from the body (from the body’s sweat glands). It is a crucial bodily function for regulating temperature, and the cooling effect occurs when sweat evaporates from the skin, absorbing excess heat from the body to do so. If a river releases liquid—oil and tar—isn’t it just to mix with the rest of the water again? Actually—some light oils can evaporate in large bodies of water, if the water is warm and the surface area is large. But it is clearly heavy oils that are meant (like crude oil or motor oil), particularly with tar following. The river sweating here and human sweating remain the same in the most basic sense: both are a function to aid the source, a kind of homeostatic regulation. Contrastingly, the river sweating functions to rid of waste—explicitly material, but probably extending to moral, spiritual, emotional. Unfortunately, this effort is doomed from the start—in a horribly looping, muddled way in that it goes round and round with the oil and tar being supposedly somewhat separated from the rest of the water, or perhaps consolidated, just to mix in again. This process was doomed from the start, but the river wasn’t, with the arrival of the oil and tar. Their presence is obtrusively unnatural. This must be remembered as the following phrases wash over with their sense of predetermination/overarching control: “The barges drift / With the turning tide…sails…swing…The barges wash / Drifting logs…”

What jumped out to me as mirroring this futile process of mixing was the lines “Weialala leia / Wallala leialala”—repeated twice, with the third iteration being just “la la.” In looking back at some past annotations, I noted that Jeannie ’25 made a comment on how “Weialala leia” can be read as a wail. In fact, when attempting to read it out loud, it sounded as though I was reciting a string of jumbled duplications of the word “wail,” and as one, it definitely sounded as a wail. This word is what the letters form, mixed around—but as more and more is added distortion grows, not clarity. In Götterdämmerung, this wailing call is for Siegfried, to return the ring, and thus the Rhine-gold. While initially appearing successful as Siegried enters right after the first call, subsequent ones fall upon deaf ears—along with the rest of the words of the Rhine Daughters. It is almost as though between when the words leave their mouths and when they reach the ears of Siegried—or the reader—there is a warping. The Rhine Daughters’ final “La! la!” feels a dwindling final attempt with recognition of the futility—the “w” is gone—or perhaps marks an even greater warping. Eliot exaggerates this further, with his third repetition being just “la la.” All of this seems to me to very much connect to female voice in the poem—particularly the line “And still she cried, and still the world pursues, / ‘Jug Jug’ to dirty ears” in “A Game of Chess.”

This reminds of last class where we talked about the achievement gap and how it should be reframed as an opportunity gap. Oftentimes the lived experiences of race and racism intersect with issues of class and access to education, and these performance evaluations which decide the level of education a student should receive fail to take into account the lived experiences of students, often limiting access to black and brown students. My high school was framed as "academically rigorous" and even though it was public, there was an admission system which was made based on applications and grades from middle school. I always found it wrong that there was an admission system, because I thought that a student's grades and ability to put together an application should not decide whether or not they had access to this academically rigorous education. While my school prided itself on its diversity, this admission system limited access to many students of color who didn't attend a highly-funded middle school where there were opportunities to thrive. This exposes a form of institutionalized racism within the school district.

Through personal experiences, I think this can end one of several ways. Students are able to find a group of students who look lime them and grow a community where theyre comfortable. They change themselves to fit into other groups despite it not being something they identify with, and at times start hating their own. And, there are fortunately some who are able to find friends who don't all have to look like them, friends who make them feel welcomed. Everyone's situation is different, and as mentioned, the need to protect oneself is understandable.

The contrast pin attitudes towards two different individuals is astounding, especially given the unknown circumstances which led the individual to get there. This text highlights how many people have a subconscious racial animus which leads them to stereotype without noticing thats what theyre doing. By only praising young black men who are good at sports it sets a stereotype that that is all they can be good at. Everyone should be praised for their talents while acknowledging that is not their only potential.

This students situation explains why many don't want to continue into higher education. Many feel isolated or as if they dont belong because they are the only person who looks like them in an advanced class. Even in higher education, I have personally felt like I dont belong because of the overwhelming population that does not look like me. Like the student mentioned, it does feel odd, but it is also something one has to be able to continue to work through to ensure the effort put into our education is worth it..

I think this stems from minor students e brain their race and identity. Why I personallyy do think I have explore my racial and ethnic identity, I was mainly due to the fact that I had to get comfortable being ethnically and racially different around other ethnic/racial groups. Many students who pursue a higher education and have to live in a place they are not used to are likely to experience a similar situation. Although I am sure there are other reasons for racial exploration, my personal experience make be think this could be true as well.

This is true for a lot of minority students who more away from home for the first time to pursue higher education. I think it is a subconscious actions, done to make one feel more welcome and at home. Minority students like myself are likely to gravitate towards others who remind us from home because it make up for the fact we miss our homes. Although this causes a divide, it also forms relationships between who otherwise would feel like imposters.

At a time when young people are actively forming their identities, the approval or disapproval of peers can shape how they express themselves not just socially, but culturally and racially. This pressure isn’t only about fitting in; it’s about aligning with a shared sense of belonging and resisting narratives imposed by the dominant culture.

This moment shows how identity can be activated by external forces rather than personal choice. As a child, David doesn’t think of race as central to who he is, but that changes once society begins to treat it as significant. His sense of self evolves not because his identity itself shifts, but because the social gaze sharpens, making race something he can’t ignore.

As students begin to define who they are, shared experiences like race, culture, or community can become central anchors in that process. It’s less about exclusion and more about finding spaces where one’s identity is affirmed and understood, which can explain why visible racial groupings emerge during adolescence.

ბლოგი შეეხება შვეიცარიაში დაგეგმილ პარკ ადულას ეროვნული პარკის პროექტს და მის ჩავარდნას, რაც ნათლად ასახავს, როგორ შეიძლება ფედერალიზმმა ხელი შეუშალოს ბიომრავალფეროვნების დაცვას. ავტორი აანალიზებს, როგორ იქცა ბუნების დაცვის ინიციატივა ადგილობრივი მოსახლეობის მხრიდან წინააღმდეგობის ობიექტად. ძირითადი მიზეზებია მოსახლეობის შიში ტრადიციული თავისუფლებების დაკარგვის, უნდობლობა მმართველი სტრუქტურების მიმართ და განცდა, რომ ლანდშაფტი უკვე საკმარისად დაცულია.

ბლოგი მკაფიოდ აჩვენებს, რომ მხოლოდ ეკოლოგიური მიზნების დასახვა არასაკმარისია — ბუნების დაცვის პოლიტიკამ წარმატებას მაშინ აღწევს, როცა ის თანხვედრაშია სოციალური, ეკონომიკური და კულტურული ინტერესებთან. ჩემი პოზიციაა, რომ აუცილებელია სახელმწიფო და ადგილობრივი ინტერესების დაბალანსება. ბიომრავალფეროვნების დაცვა უნდა ეფუძნებოდეს არა მხოლოდ მეცნიერებასა და გარემოსდაცვით მიზნებს, არამედ მოსახლეობის მონაწილეობასა და ნდობაზე დამყარებულ პროცესს. წინააღმდეგ შემთხვევაში, ყველაზე კეთილი განზრახვებიც კი წარუმატებელი დარჩება.

for - adjacency - memory and imagination - episodic memory

for definition - Mental Time Travel - Neither Time nor Travel may be defining characteristics of Mental Time Travel - Mental rendering of experience may be the defining characteristic

for - claim - Mental Time Travel into the past and future are instantiations of one simulation system

for - adjacency - past - future - Michael Levin - memories - goal-seeking - Michael Levin's research validates that memories serve the role of goak-seeking - This is another way of saying that the past plays a critical role in the future

for - definition - Mental Time Travel - ability to look at the past and the future

for - adjacency - memory - imagination - the same - claim - memory - imagination - the same

for - researcher - neuroscience - memory - perception - imagination - to - Mental Time Travel? - Donna Rose Addis - https://hyp.is/wqV4gKdkEfCRZGPrIOjeOA/utoronto.scholaris.ca/server/api/core/bitstreams/3232f1fb-ed19-4614-9dd5-648c4d443629/content

for - Memory Theory of Consciousness - MToC - definition - Memory Theory of Consciousness - temporally extended, multimodal representations - must be integrated within a unified subjectivity for experience to be coherent - unapack - MToC - unpack - Memory Theory of Consciousness - temporally extended, multimodal representations - multiple sense inputs associated with an event - We could think about it from the perspective of Thousand Brain Theory and cortical columns integrating sense inputs - Do these create memory structures? - Those memory structures must be salient to goal-seeking activity, especially for fitness and survival of the organism

question - memory - evolution - goal-seeking - Is it possible that consciousness emerged early on in our species evolutionary history in the context of memories of multimodal sensory structures that help us achieve goal-seeking activity? - Then extra affordances of memory and consciousness could have evolved and diversified into a wide variety of non-traditional goal-seeking behaviors.

for - Kant - memory

I totally agree computer are algorithms and there are rules that come along with it.

Technology is definitely different and make then it was not advanced as it is today but i lowkey wish technology didn’t get so advanced!!

He took a risk to give up things to make web do make Technology better and so that we can have web browsers 😝

Love how is Creation expanded!!! 👏🏽👏🏽

AI is taking over and honestly it makes me MADDD ! ![(https://share.google/PsjCvwOnCoRVISoeg)

• Planning : what your are going to do
• Budget : How you are going to to it, quantifying the plan
• Control : planning + evaluation and feedback

By assuming Asian-American students will succeed without support, educators may unintentionally ignore their individual learning needs. This reflects a larger issue: stereotypes whether negative or flattering flatten complex identities into a single narrative, often silencing the very students they claim to praise.

I believe that you have included them according to the slides at least adsorption

?

This is definitely something I have referenced in my career, that almost all disruptive or behavioral issues are always developmentally appropriate for children. I do wonder if there is a specific line on when it may pass into inappropriate and how to handle that in the best way. My co-workers and I never take anything personally and always use our experience and knowledge to understand that children are just children and are being developmentally appropriate for their age, probably 99 percent of the time. It's surprising to me that there is any situation that a child's behavior may be considered developmentally inappropriate as I can't say I have had to experience that first hand.

Attribution over referencing

Functional literacy: How does AI work?

Ethical literacy: How do we navigate the ethical issues of AI?

Rhetorical literacy: How do we use natural and AI-generated language to achieve our goals?

Pedagogical literacy: How do we use AI to enhance teaching and learning?

Encouraging AI literacy for educators not just students. Potential idea for a CFE Repository...

no idea what to do with this but it seems significant

good point

this is an interesting way of explaining it and explaining ease of similar forms with regular rules

so this has lexical sets then turned into rules

useful section for essay 1 about the difference and consequences for the different models

Important. As with an study or trial, it needs to be replicated in all of the educational settings.

These are key points I would share with students who are hesitant to publicly share. A delicate balance to be sure, you want the student to have autonomy and agency, moreso to encourage the student to feel confident about their work and sharing it.

Combatting stereotypes also requires the educator to do a bit of soul-searching - it is imperative that we (in pursuit of implementing social justices vis-a-vis OER) recognize and identify our own biases to ensure minimal overlap when selecting resources.

Peer collaboration is critical! We've all seen students become just a bit more [unintentionally smize] engaged when encountering a new learning experience after interacting with peers - like when they ask each other questions instead of the instructor! I am literal application of this theory. I struggled with setting up my edublog for the students - until I reached out to colleagues.

I find that my students struggle with the concept of privacy - as they've been immersed in social media (even if they don't have accounts/have limited interactions) and virtual interactions (social, learning, gaming etc.) It's my observation that giving students options on how/if they go "public" can be very empowering for them. Such as if they are not fully confident in their work, or already feel marginalized by the topic, text, or as a demographic at the institution.

Gregor Samsa

moral of the story

instead of a statutory regime, what tort liability permits is for discovery of realized harms

self

open self link to see it via.hypothes.is that will et you see the embedded screenshots/image

video

eLife Assessment

This high-N, multi-task study offers a comprehensive examination of rhythmicity in behavioral performance during listening. It presents a valuable set of findings that reveal task- and ear-specific effects, challenging the notion of a universal rhythmicity in auditory perception. The evidence is solid and the work is likely to be of significant interest to behavioral and cognitive scientists focused on perception and neural oscillations.

Reviewer #1 (Public review):

Summary:

This paper presents results from four independent experiments, each of them testing for rhythmicity in auditory perception. The authors report rhythmic fluctuations in discrimination performance at frequencies between 2 and 6 Hz. The exact frequency depends on the ear and experimental paradigm, although some frequencies seem to be more common than others.

Strengths:

The first sentence in the abstract describes the state of the art perfectly: "Numerous studies advocate for a rhythmic mode of perception; however, the evidence in the context of auditory perception remains inconsistent". This is precisely why the data from the present study is so valuable. This is probably the study with the highest sample size (total of > 100 in 4 experiments) in the field. The analysis is very thorough and transparent, due to the comparison of several statistical approaches and simulations of their sensitivity. Each of the experiments differs from the others in a clearly defined experimental parameter, and the authors test how this impacts auditory rhythmicity, measured in pitch discrimination performance (accuracy, sensitivity, bias) of a target presented at various delays after noise onset.

Weaknesses:

The authors find that the frequency in auditory perception changes between experiments. Possible reasons for such differences are described, but they remain difficult to interpret, as it is unclear whether they merely reflect some natural variability (independent of experimental parameters) or are indeed driven by the specific experimental paradigm (and therefore replicable).

Therefore, it remains to be shown whether there is any systematic pattern in the results that allows conclusions about the roles of different frequencies.

Reviewer #2 (Public review):

Summary:

The current study aims to shed light on why previous work on perceptual rhythmicity has led to inconsistent results. They propose that the differences may stem from conceptual and methodological issues. In a series of experiments, the current study reports perceptual rhythmicity in different frequency bands that differ between different ear stimulations and behavioral measures. The study suggests challenges regarding the idea of universal perceptual rhythmicity in hearing.

Strengths:

The study aims to address differences observed in previous studies about perceptual rhythmicity. This is important and timely because the existing literature provides quite inconsistent findings. Several experiments were conducted to assess perceptual rhythmicity in hearing from different angles. The authors use sophisticated approaches to address the research questions. The manuscript has greatly improved after the revision.

Weaknesses:

Additional variance: In several experiments, a fixation cross preceded - at a variable interval - the onset of the background noise that aimed to reset the phase of an ongoing oscillation. There is the chance that the fixation cross also resets the phase, potentially adding variance to the data. In addition, the authors used an adaptive procedure during the experimental blocks such that the stimulus intensity was adjusted throughout. There is good reason for doing so, but it means that correctly identified/discriminated targets will on average have a greater stimulus intensity. This may add variance to the data. These two aspects may potentially contribute to the observation of weak perceptual rhythmicity.

Figures: The text in Figures 4 and 6 is small. I think readers would benefit from a larger font size. Moreover, Figure 1A is not very intuitive. Perhaps it could be made clearer. The new Figure 5 was not discussed in the text. I wonder whether analyses with traditional t-tests could be placed in the supplements.

50% significant samples: The authors consider 50% of significant bootstrapped samples robust. For example: "This revealed that the above‐mentioned effects prevail for at least 50% of the simulated experiments, corroborating their robustness within the participant sample". Many of the effects have even lower than 50% of significant samples. It is a matter of opinion of what is robust or not, but I think combined with the overall variable nature of the effects in different frequency bands and ears etc. leaves more the impression that the effects are not very robust. I think the authors state it correctly in the last sentence of the first paragraph of the discussion: "At the same time the prevalence of significant effects in random samples of participants were mostly below 50%, raising questions as to the ubiquity of such effects." I think the authors should update the abstract in this regard to avoid that readers who only read the abstract get the wrong impression about the robustness of the effects. It is not clear to me if the same study (using the same conditions) was done in a different lab that the results would come out similarly to the results reported here.

Reviewer #3 (Public review):

Summary:

The finding of rhythmic activity in the brain has for a long time engendered the theory of rhythmic modes of perception, that humans might oscillate between improved and worse perception depending on states of our internal systems. However, experiments looking for such modes have resulted in conflicting findings, particularly in those where the stimulus itself is not rhythmic. This paper seeks to take a comprehensive look at the effect and various experimental parameters which might generate these competing findings: in particular, the presentation of the stimulus to one ear or the other, the relevance of motor involvement, attentional demands, and memory: each of which are revealed to effect the consistency of this rhythmicity.

The need the paper attempts to resolve is a critical one for the field. However, as presented, I remain unconvinced that the data would not be better interpreted as showing no consistent rhythmic mode effect.

Strengths:

The paper is strong in its experimental protocol and its comprehensive analysis which seeks to compare effects across several analysis types and slight experiment changes to investigate which parameters could effect the presence or absence of an effect of rhythmicity. The prescribed nature of its hypotheses and its manner to set out to test them is very clear which allows for a straightforward assessment of its results

Weaknesses:

The papers cited to justify a rhythmic mode are largely based on the processing of rhythmic stimuli. The authors assume the rhythmic mode to be the general default but its not so clear to me why this would be so. The task design seems better suited to a continuous vigilance mode task.

Secondly, the analysis to detect a "rhythmic mode", assumes a total phase rest at noise onset which is highly implausible given standard nonlinear dynamical analysis of oscillator performance. It's not clear that a rhythmic mode (should it be applied in this task) would indeed generate a consistent phase as the analysis searches for.

Thirdly, the number of statistical tests used here make trusting any single effect quite difficult and very few of the effects replicate more than once. I think the better would be interpreted as not confirming evidence for rhythmic mode processing in the ears.

Comments on revised version:

No further comments. The paper has much of the same issues that I expressed in the initial review but I don't think they can be addressed without a replication study which I appreciate is not always plausible.

Author response:

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

Reviewer #1 (Public review):

Summary:

This paper presents results from four independent experiments, each of which tests for rhythmicity in auditory perception. The authors report rhythmic fluctuations in discrimination performance at frequencies between 2 and 6 Hz. The exact frequency depends on the ear and experimental paradigm, although some frequencies seem to be more common than others.

Strengths:

The first sentence in the abstract describes the state of the art perfectly: "Numerous studies advocate for a rhythmic mode of perception; however, the evidence in the context of auditory perception remains inconsistent". This is precisely why the data from the present study is so valuable. This is probably the study with the highest sample size (total of > 100 in 4 experiments) in the field. The analysis is very thorough and transparent, due to the comparison of several statistical approaches and simulations of their sensitivity. Each of the experiments differs from the others in a clearly defined experimental parameter, and the authors test how this impacts auditory rhythmicity, measured in pitch discrimination performance (accuracy, sensitivity, bias) of a target presented at various delays after noise onset.

Weaknesses:

(1) The authors find that the frequency of auditory perception changes between experiments. I think they could exploit differences between experiments better to interpret and understand the obtained results. These differences are very well described in the Introduction, but don't seem to be used for the interpretation of results. For instance, what does it mean if perceptual frequency changes from between- to within-trial pitch discrimination? Why did the authors choose this experimental manipulation? Based on differences between experiments, is there any systematic pattern in the results that allows conclusions about the roles of different frequencies? I think the Discussion would benefit from an extension to cover this aspect.

We believe that interpreting these differences remains difficult and a precise, detailed (and possibly mechanistic) interpretation is beyond the goal of the present study. The main goal of this study was to explore the consistency and variability of effects across variations of the experimental design and samples of participants. Interpreting specific effects, e.g. at particular frequencies, would make sense mostly if differences between experiments have been confirmed in a separate reproduction. Still, we do provide specific arguments for why differences in the outcome between different experiments, e.g. with and without explicit trial initialization by the participants, could be expected. See lines 91ff in the introduction and 786ff in the discussion.

(2) The Results give the impression of clear-cut differences in relevant frequencies between experiments (e.g., 2 Hz in Experiment 1, 6 Hz in Exp 2, etc), but they might not be so different. For instance, a 6 Hz effect is also visible in Experiment 1, but it just does not reach conventional significance. The average across the three experiments is therefore very useful, and also seems to suggest that differences between experiments are not very pronounced (otherwise the average would not produce clear peaks in the spectrum). I suggest making this point clearer in the text.

We have revised the conclusions to note that the present data do not support clear cut differences between experiments. For this reason we also refrain from detailed interpretations of specific effects, as suggested by this reviewer in point 1 above.

(3) I struggle to understand the hypothesis that rhythmic sampling differs between ears. In most everyday scenarios, the same sounds arrive at both ears, and the time difference between the two is too small to play a role for the frequencies tested. If both ears operate at different frequencies, the effects of the rhythm on overall perception would then often cancel out. But if this is the case, why would the two ears have different rhythms to begin with? This could be described in more detail.

This hypothesis was not invented by us, but in essence put forward in previous work. The study by Ho et al. CurrBiol 2017 has reported rhythmic effects at different frequencies in the left and right ears, and we here tried to reproduce these effects. One could speculate about an ear-difference based on studies reporting a right-ear advantage in specific listening tasks, and the idea that different time scales of rhythmic brain activity may be specifically prevail in the left and right cortical hemispheres; hence it does not seem improbable that there could be rhythmic effects in both ears at different frequencies. We note this in the introduction, l. 65ff.

Reviewer #2 (Public review):

Summary:

The current study aims to shed light on why previous work on perceptual rhythmicity has led to inconsistent results. They propose that the differences may stem from conceptual and methodological issues. In a series of experiments, the current study reports perceptual rhythmicity in different frequency bands that differ between different ear stimulations and behavioral measures.

The study suggests challenges regarding the idea of universal perceptual rhythmicity in hearing.

Strengths:

The study aims to address differences observed in previous studies about perceptual rhythmicity. This is important and timely because the existing literature provides quite inconsistent findings. Several experiments were conducted to assess perceptual rhythmicity in hearing from different angles. The authors use sophisticated approaches to address the research questions.

Weaknesses:

(1) Conceptional concerns:

The authors place their research in the context of a rhythmic mode of perception. They also discuss continuous vs rhythmic mode processing. Their study further follows a design that seems to be based on paradigms that assume a recent phase in neural oscillations that subsequently influence perception (e.g., Fiebelkorn et al.; Landau & Fries). In my view, these are different facets in the neural oscillation research space that require a bit more nuanced separation. Continuous mode processing is associated with vigilance tasks (work by Schroeder and Lakatos; reduction of low frequency oscillations and sustained gamma activity), whereas the authors of this study seem to link it to hearing tasks specifically (e.g., line 694). Rhythmic mode processing is associated with rhythmic stimulation by which neural oscillations entrain and influence perception (also, Schroeder and Lakatos; greater low-frequency fluctuations and more rhythmic gamma activity). The current study mirrors the continuous rather than the rhythmic mode (i.e., there was no rhythmic stimulation), but even the former seems not fully fitting, because trials are 1.8 s short and do not really reflect a vigilance task. Finally, previous paradigms on phase-resetting reflect more closely the design of the current study (i.e., different times of a target stimulus relative to the reset of an oscillation). This is the work by Fiebelkorn et al., Landau & Fries, and others, which do not seem to be cited here, which I find surprising. Moreover, the authors would want to discuss the role of the background noise in resetting the phase of an oscillation, and the role of the fixation cross also possibly resetting the phase of an oscillation. Regardless, the conceptional mixture of all these facets makes interpretations really challenging. The phase-reset nature of the paradigm is not (or not well) explained, and the discussion mixes the different concepts and approaches. I recommend that the authors frame their work more clearly in the context of these different concepts (affecting large portions of the manuscript).

Indeed, the paradigms used here and in many similar previous studies incorporate an aspect of phase-resetting, as the presentation of a background noisy may effectively reset ongoing auditory cortical processes. Studies trying to probe for rhythmicity in auditory perception in the absence any background noise have not shown any effect (Zoefel and Heil, 2013), perhaps because the necessary rhythmic processes along auditory pathways are only engaged when some sound is present. We now discuss these points, and also acknowledge the mentioned studies in the visual system; l. 57.

(2) Methodological concerns:

The authors use a relatively unorthodox approach to statistical testing. I understand that they try to capture and characterize the sensitivity of the different analysis approaches to rhythmic behavioral effects. However, it is a bit unclear what meaningful effects are in the study. For example, the bootstrapping approach that identifies the percentage of significant variations of sample selections is rather descriptive (Figures 5-7). The authors seem to suggest that 50% of the samples are meaningful (given the dashed line in the figure), even though this is rarely reached in any of the analyses. Perhaps >80% of samples should show a significant effect to be meaningful (at least to my subjective mind). To me, the low percentage rather suggests that there is not too much meaningful rhythmicity present. 

We note that there is no clear consensus on what fraction of experiments should be expected or how this way of quantifying effects should be precisely valued (l. 441ff). However, we now also clearly acknowledge in the discussion that the effective prevalence is not very high (l. 663).

I suggest that the authors also present more traditional, perhaps multi-level, analyses: Calculation of spectra, binning, or single-trial analysis for each participant and condition, and the respective calculation of the surrogate data analysis, and then comparison of the surrogate data to the original data on the second (participant) level using t-tests. I also thought the statistical approach undertaken here could have been a bit more clearly/didactically described as well.

We here realize that our description of the methods was possibly not fully clear. We do follow the strategy as suggested by this reviewer, but rather than comparing actual and surrogate data based on a parametric t-test, we compare these based on a non-parametric percentile-based approach. This has the advantage of not making specific (and possibly not-warranted) assumptions about the distribution of the data. We have revised the methods to clarify this, l. 332ff. 

The authors used an adaptive procedure during the experimental blocks such that the stimulus intensity was adjusted throughout. In practice, this can be a disadvantage relative to keeping the intensity constant throughout, because, on average, correct trials will be associated with a higher intensity than incorrect trials, potentially making observations of perceptual rhythmicity more challenging. The authors would want to discuss this potential issue. Intensity adjustments could perhaps contribute to the observed rhythmicity effects. Perhaps the rhythmicity of the stimulus intensity could be analyzed as well. In any case, the adaptive procedure may add variance to the data.

We have added an analysis of task difficulty to the results (new section “Effects of adaptive task difficulty“) to address this. Overall we do not find systematic changes in task difficulty across participants for most of the experiments, but for sure one cannot rule out that this aspect of the design also affects the outcomes.  Importantly, we relied on an adaptive task difficulty to actually (or hopefully) reduce variance in the data, by keeping the task-difficulty around a certain level. Give the large number of trials collected, not using such an adaptive produce may result in performance levels around chance or near ceiling, which would make impossible to detect rhythmic variations in behavior. 

Additional methodological concerns relate to Figure 8. Figures 8A and C seem to indicate that a baseline correction for a very short time window was calculated (I could not find anything about this in the methods section). The data seem very variable and artificially constrained in the baseline time window. It was unclear what the reader might take from Figure 8.

This figure was intended mostly for illustration of the eye tracking data, but we agree that there is no specific key insight to be taken from this. We removed this. 

Motivation and discussion of eye-movement/pupillometry and motor activity: The dual task paradigm of Experiment 4 and the reasons for assessing eye metrics in the current study could have been better motivated. The experiment somehow does not fit in very well. There is recent evidence that eye movements decrease during effortful tasks (e.g., Contadini-Wright et al. 2023 J Neurosci; Herrmann & Ryan 2024 J Cog Neurosci), which appears to contradict the results presented in the current study. Moreover, by appealing to active sensing frameworks, the authors suggest that active movements can facilitate listening outcomes (line 677; they should provide a reference for this claim), but it is unclear how this would relate to eye movements. Certainly, a person may move their head closer to a sound source in the presence of competing sound to increase the signal-to-noise ratio, but this is not really the active movements that are measured here. A more detailed discussion may be important. The authors further frame the difference between Experiments 1 and 2 as being related to participants' motor activity. However, there are other factors that could explain differences between experiments. Self-paced trials give participants the opportunity to rest more (inter-trial durations were likely longer in Experiment 2), perhaps affecting attentional engagement. I think a more nuanced discussion may be warranted.

We expanded the motivation of why self-pacing trials may effectively alter how rhythmic processes affect perception, and now also allude to attention and expectation related effects (l. 786ff). Regarding eye movements we now discuss the results in the light of the previously mentioned studies, but again refrain from a very detailed and mechanistic interpretation (l. 782).

Discussion:

The main data in Figure 3 showed little rhythmicity. The authors seem to glance over this fact by simply stating that the same phase is not necessary for their statistical analysis. Previous work, however, showed rhythmicity in the across-participant average (e.g., Fiebelkorn's and similar work). Moreover, one would expect that some of the effects in the low-frequency band (e.g., 2-4 Hz) are somewhat similar across participants. Conduction delays in the auditory system are much smaller than the 0.25-0.5 s associated with 2-4 Hz. The authors would want to discuss why different participants would express so vastly different phases that the across-participant average does not show any rhythmicity, and what this would mean neurophysiologically.

We now discussion the assumptions and implications of similar or distinct phases of rhythmic processes within and between participants (l. 695ff). In particular we note that different origins of the underlying neurophysiological processes eventually may suggest that such assumptions are or a not warranted.  

An additional point that may require more nuanced discussion is related to the rhythmicity of response bias versus sensitivity. The authors could discuss what the rhythmicity of these different measures in different frequency bands means, with respect to underlying neural oscillations.

We expanded discussion to interpret what rhythmic changes in each of the behavioral metric could imply (l. 706ff).

Figures:

Much of the text in the figures seems really small. Perhaps the authors would want to ensure it is readable even for those with low vision abilities. Moreover, Figure 1A is not as intuitive as it could be and may perhaps be made clearer. I also suggest the authors discuss a bit more the potential monoaural vs binaural issues, because the perceptual rhythmicity is much slower than any conduction delays in the auditory system that could lead to interference.

We tried to improve the font sizes where possible, and discuss the potential monaural origins as suggested by other reviewers. 

Reviewer #3 (Public review):

Summary:

The finding of rhythmic activity in the brain has, for a long time, engendered the theory of rhythmic modes of perception, that humans might oscillate between improved and worse perception depending on states of our internal systems. However, experiments looking for such modes have resulted in conflicting findings, particularly in those where the stimulus itself is not rhythmic. This paper seeks to take a comprehensive look at the effect and various experimental parameters which might generate these competing findings: in particular, the presentation of the stimulus to one ear or the other, the relevance of motor involvement, attentional demands, and memory: each of which are revealed to effect the consistency of this rhythmicity.

The need the paper attempts to resolve is a critical one for the field. However, as presented, I remain unconvinced that the data would not be better interpreted as showing no consistent rhythmic mode effect. It lacks a conceptual framework to understand why effects might be consistent in each ear but at different frequencies and only for some tasks with slight variants, some affecting sensitivity and some affecting bias.

Strengths:

The paper is strong in its experimental protocol and its comprehensive analysis, which seeks to compare effects across several analysis types and slight experiment changes to investigate which parameters could affect the presence or absence of an effect of rhythmicity. The prescribed nature of its hypotheses and its manner of setting out to test them is very clear, which allows for a straightforward assessment of its results

Weaknesses:

There is a weakness throughout the paper in terms of establishing a conceptual framework both for the source of "rhythmic modes" and for the interpretation of the results. Before understanding the data on this matter, it would be useful to discuss why one would posit such a theory to begin with. From a perceptual side, rhythmic modes of processing in the absence of rhythmic stimuli would not appear to provide any benefit to processing. From a biological or homeostatic argument, it's unclear why we would expect such fluctuations to occur in such a narrow-band way when neither the stimulus nor the neurobiological circuits require it.

We believe that the framework for why there may be rhythmic activity along auditory pathways that shapes behavioral outcomes has been laid out in many previous studies, prominently here (Schroeder et al., 2008; Schroeder and Lakatos, 2009; Obleser and Kayser, 2019). Many of the relevant studies are cited in the introduction, which is already rather long given the many points covered in this study. 

Secondly, for the analysis to detect a "rhythmic mode", it must assume that the phase of fluctuations across an experiment (i.e., whether fluctuations are in an up-state or down-state at onset) is constant at stimulus onset, whereas most oscillations do not have such a total phase-reset as a result of input. Therefore, some theoretical positing of what kind of mechanism could generate this fluctuation is critical toward understanding whether the analysis is well-suited to the studied mechanism.

In line with this and previous comments (by reviewer 2) we have expanded the discussion to consider the issue of phase alignment (l. 695ff). 

Thirdly, an interpretation of why we should expect left and right ears to have distinct frequency ranges of fluctuations is required. There are a large number of statistical tests in this paper, and it's not clear how multiple comparisons are controlled for, apart from experiment 4 (which specifies B&H false discovery rate). As such, one critical method to identify whether the results are not the result of noise or sample-specific biases is the plausibility of the finding. On its face, maintaining distinct frequencies of perception in each ear does not fit an obvious conceptual framework.

Again this point was also noted by another reviewer and we expanded the introduction and discussion in this regard (l. 65ff).

Reviewer #1 (Recommendations for the authors):

(1) An update of the AR-surrogate method has recently been published (https://doi.org/10.1101/2024.08.22.609278). I appreciate that this is a lot of work, and it is of coursee up to the authors, but given the higher sensitivity of this method, it might be worth applying it to the four datasets described here.

Reading this article we note that our implementation of the AR-surrogate method was essentially as suggested here, and not as implemented by Brookshire. In fact we had not realized that Brookshire had apparently computed the spectrum based on the group-average data. As explained in the Methods section, as now clarified even better, we compute for each participant the actual spectrum of this participant’s data, and a set of surrogate spectra. We then perform a group-average of both to compute the p-value of the actual group-average based on the percentile of the distribution of surrogate averages. This send step differs from Harris & Beale, which used a one-sided t-test. The latter is most likely not appropriate in a strict statistical sense, but possibly more powerful for detecting true results compared to the percentile-based approach that we used (see l. 332ff).

(2) When results for the four experiments are reported, a reminder for the reader of how these experiments differ from each other would be useful.

We have added this in the Results section.

"considerable prevalence of differences around 4Hz, with dual‐task requirements leading to stronger rhythmicity in perceptual sensitivity". There is a striking similarity to recently published data (https://doi.org/10.1101/2024.08.10.607439 ) demonstrating a 4-Hz rhythm in auditory divided attention (rather than between modalities as in the present case). This could be a useful addition to the paragraph.

We have added a reference to this preprint, and additional previous work pointing in the same direction mentioned in there.  

(3) There are two typos in the Introduction: "related by different from the question", and below, there is one "presented" too much.

These have been fixed.

Reviewer #3 (Recommendations for the authors):

My major suggestion is that these results must be replicated in a new sample. I understand this is not simple to do and not always possible, but at this point, no effect is replicated from one experiment to the next, despite very small changes in protocol (especially experiment 1 vs 2). It's therefore very difficult to justify explaining the different effects as real as opposed to random effects of this particular sample. While the bootstrapping effects show the level of consistency of the effect within the sample studied, it can not be a substitute for a true replication of the results in a new sample.

We agree that only an independent replication can demonstrate the robustness of the results. We do consider experiment 1 a replication test of Ho et al. CurrBiol 2017, which results in different results than reported there. But more importantly, we consider the analysis of ‘reproducibility’ by simulating participant samples a key novelty of the present work, and want to emphasize this over the within-study replication of the same experiment.  In fact, in light of the present interpretation of the data, even a within-study replication would most likely not offer a clear-cut answer. 

As I said in the public review, the interpretation of the results, and of why perceptual cycles in arhythmic stimuli could be a plausible theory to begin with, is lacking. A conceptual framework would vastly improve the impact and understanding of the results.

We tried to strengthen the conceptual framework in the introduction. We believe that this is in large provided by previous work, and the aim of the present study was to explore the robustness of effects and not to suggest and discover novel effects. 

Minor comments:

(1) The authors adapt the difficulty as a function of performance, which seems to me a strange choice for an experiment that is analyzing the differences in performance across the experiment. Could you add a sentence to discuss the motivation for this choice?

We now mention the rationale in the Methods section and in a new section of the Results. There we also provide additional analyses on this parameter.

(2) The choice to plot the p-values as opposed to the values of the actual analysis feels ill-advised to me. It invites comparison across analyses that isn't necessarily fair. It would be more informative to plot the respective analysis outputs (spectral power, regression, or delta R2) and highlight the windows of significance and their overlap across analyses. In my opinion, this would be more fair and accurate depiction of the analyses as they are meant to be used.

We do disagree. As explained in the Methods (l. 374ff): “(Showing p-values) … allows presenting the results on a scale that can be directly compared between analysis approaches, metrics, frequencies and analyses focusing on individual ears or the combined data. Each approach has a different statistical sensitivity, and the underlying effect sizes (e.g. spectral power) vary with frequency for both the actual data and null distribution. As a result, the effect size reaching statistical significance varies with frequency, metrics and analyses.” 

The fact that the level of power (or R2 or whatever metric we consider) required to reach significance differs between analyses (one ear, both ears), metrics (d-prime, bias, RT) and between analyses approaches makes showing the results difficult, as we would need a separate panel for each of those. This would multiply the number of panels required e.g. for Figure 4 by 3, making it a figure with 81 axes. Also neither the original quantities of each analysis (e.g. spectral power) nor the p-values that we show constitute a proper measure of effect size in a statistical sense. In that sense, neither of these is truly ideal for comparing between analyses, metrics etc. 

We do agree thought that many readers may want to see the original quantification and thresholds for statistical significance. We now show these in an exemplary manner for the Binned analysis of Experiment 1, which provides a positive result and also is an attempt to replicate the findings by  Ho et al 2017. This is shown in new Figure 5. 

(3) Typo in line 555 (+ should be plus minus).

(4) Typo in line 572: "Comparison of 572 blocks with minus dual task those without"

(5) Typo in line 616: remove "one".

(6) Line 666 refers to effects in alpha band activity, but it's unclear what the relationship is to the authors' findings, which peak around 6 Hz, lower than alpha (~10 Hz).

(7) Line 688 typo, remove "amount of".

These points have been addressed.  

(8) Oculomotor effect that drives greater rhythmicity at 3-4 Hz. Did the authors analyze the eye movements to see if saccades were also occurring at this rate? It would be useful to know if the 3-4 Hz effect is driven by "internal circuitry" in the auditory system or by the typical rate of eye movement.

A preliminary analysis of eye movement data was in previous Figure 8, which was removed on the recommendation of another review.  This showed that the average saccade rate is about 0.01 saccade /per trial per time bin, amounting to on average less than one detected saccade per trial. Hence rhythmicity in saccades is unlikely to explain rhythmicity in behavioral data at the scale of 34Hz. We now note this in the Results.

Obleser J, Kayser C (2019) Neural Entrainment and Attentional Selection in the Listening Brain. Trends Cogn Sci 23:913-926.

Schroeder CE, Lakatos P (2009) Low-frequency neuronal oscillations as instruments of sensory selection. Trends Neurosci 32:9-18.

Schroeder CE, Lakatos P, Kajikawa Y, Partan S, Puce A (2008) Neuronal oscillations and visual amplification of speech. Trends Cogn Sci 12:106-113.

Zoefel B, Heil P (2013) Detection of Near-Threshold Sounds is Independent of EEG Phase in Common Frequency Bands. Front Psychol 4:262.

how the first explaination is tested

one question that the existing paper asks

i did not really understand this. like the pressure of a competitive performance itself instead of things like overconfidence, risk aversion etc is what the 1st explanation measures? but aren't these things very correlated

so our task needs to be something that is categorised as a masculine domain

features of the existing paper - justifying our speculations of results (all features of existing is to help us write our final results/final written thingies w the theory parts)**

speculation for reason results of the existing paper

feature of existing paper result/existing paper methodology need to figure how to include in our study

methodology of existing paper need to figure how to take care of this in our study

result of existing paper

He who Doubts from what he sees

Will neer Believe do what you Please

If the Sun & Moon should Doubt

Theyd immediately Go out

Sacrificing control over you body for fame and bein "free"

The beginnings of Vaudville

How had tap been codified today? Has it lost it's relevance?

Were Dunn and Glover battling or learning?

May signify a change, he describes the ocean with a different intensity than the lake

Shows that he was enjoying the lake and saw it as a positive thing

There intentions that affect how someone tells the story, even if they don’t say them directly.

carefully use your critical thinking skills, maybe think to where * What kind of source it is (for example, a book, article, or website) * How up-to-date the information is * How trustworthy the publication is * Why it was created and who or what it’s meant for

Quickly looking over a book or article can help you quickly figure out what are the main ideas included, without having to read everything in detail especially if your kinda in a rush

Reasoning is more like illation

injecting the mechanical view

Must read Hobbes

there is more to Reasoning than what can be captured in discourse and Rational Arguments

DeFi integrated NFT marketplace opens doors to enhanced liquidity, NFT collateralization, and passive income opportunities. Discover how DeFi protocols can revolutionize digital asset trading and ownership. Discover the key benefits and strategies for implementing this innovation effectively. Read our blog, Build NFT Marketplace Powered by DeFi Protocols, to explore more.

eLife Assessment

The reviewers have found that this manuscript is a valuable contribution, and the evidence in support of its conclusions is mostly solid. It provides novel insights and raises interesting possibilities about the functions of an understudied histone modification within the nucleosome core; however, the data are mostly descriptive and correlative, and although this has value, it is not totally persuasive. Short of additional non-genomic experiments, a more detailed analysis of the genomic data and perhaps additional data would strengthen the conclusions. The manuscript crucially needs further antibody validation to raise confidence in the data.

Reviewer #1 (Public review):

Summary:

The authors investigate the role of H3K115ac in mouse embryonic stem cells. They report that H3K115ac localizes to regions enriched for fragile nucleosomes, CpG islands, and enhancers, and that it correlates with transcriptional activity. These findings suggest a potential role for this globular domain modification in nucleosome dynamics and gene regulation. If robust, these observations would expand our understanding of how non-tail histone modifications contribute to chromatin accessibility and transcriptional control.

Strengths:

(1) The study addresses a histone PTM in the globular domain, which is relatively unexplored compared to tail modifications.

(2) The implication of a histone PTM in fragile nucleosome localization is novel and, if substantiated, could represent a significant advance for the field.

Weaknesses:

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

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

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

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

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

Weaknesses:

(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.

(2) The association of H3K115ac with fragile nucleosomes 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.

(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.

(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.

(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.

At the very least, the authors should acknowledge these limitations and provide additional validation of antibody specificity.

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 serve as 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 the H3K115ac mark may function to destabilize nucleosomes and contribute to regulating ESC differentiation.

Weaknesses:

Additional experiments to confirm antibody specificity are needed. The authors use synthetic peptides for other markers (e.g., H3K122) to support the claim that the antibody is specific, but ChIP-ChIP assays are performed under cross-linked, non-denatured conditions, which preserve structure and epitope accessibility differently than synthetic peptides used for dot blots. Does the antibody give a single band in western blots of histones, and can the H3K115ac peptide block western and immunofluorescence signals of the antibody? Given that the antibody is a rabbit polyclonal, specificity is not a trivial consideration.

eLife Assessment

This important study establishes bathy phytochromes, a unique class of bacterial photoreceptors that respond to near-infrared light (NIR), as versatile tools for bacterial optogenetics. NIR light is a key control signal in optogenetics due to its deep tissue penetration and the ability to combine with existing red- and blue-light sensitive systems, but thus far, NIR-activated proteins have been poorly characterized. The strength of evidence is convincing, with comprehensive in vitro characterization, modular design strategies, and validation across different hosts, supporting the versatility and potential for these tools in biotechnological applications. This study should advance the fields of optogenetics and photobiology and inspire future work.

Reviewer #1 (Public review):

Summary:

This is an interesting study characterizing and engineering so-called bathy phytochromes, i.e. those that respond to near infrared (NIR) light in the ground state, for optogenetic control of bacterial gene expression. Previously, the authors have developed a structure-guided approach to functionally link several light responsive protein domains to the signaling domain of the histidine kinase FixL, which ultimately controls gene expression. Here, the authors use the same strategy to link bathy phytochrome light responsive domains to FixL, resulting in sensors of NIR light. Interestingly, they also link these bathy phytochrome light sensing domains to signaling domains from the tetrathionate-sensing SHK TtrS and the toluene-sensing SHK TodS, demonstrating generality of their protein engineering approach more broadly across bacterial two-component systems.

This is an exciting result that should inspire future bacterial sensor design. The authors go on to leverage this result to develop what is, to my knowledge, the first system for orthogonally controlling the expression of two separate genes in the same cell with NIR and Red light, a valuable contribution to the field.

Finally, the authors reveal new details of the pH-dependent photocycle of bathy phytochromes and demonstrate their sensors work in the gut- and plant-relevant strains E. coli Nissle 1917 and A. tumefaciens.

Strengths:

The experiments are well founded, well executed, and rigorous.

The manuscript is clearly written.

The sensors developed exhibit large responses to light, making them valuable tools for ontogenetic applications.

This study is a valuable contribution to photobiology and optogenetics.

Weaknesses:

As the authors note, the sensors are relatively insensitive to NIR light due to the rapid dark reversion process in bathy phytochromes. Though NIR light is generally non-phototoxic, one would expect this characteristic to be a limitation in some downstream applications where light intensities are not high (e.g. in vivo).

Though they can be multiplexed with Red light sensors, these bathy phytochrome NIR sensors are more difficult to multiplex with other commonly used light sensors (e.g. blue) due to the broad light responsivity of the Pfr state. This challenge may be overcome by careful dosing of blue light, as the authors discuss, but other bacterial NIR sensing systems with less cross-talk may be preferred in some applications.

Comments on revisions:

My concerns have been addressed.

Reviewer #2 (Public review):

In this manuscript, Meier et al. engineer a new class of light-regulated two-component systems. These systems are built using bathy-bacteriophytochromes that respond to near-infrared (NIR) light. Through a combination of genetic engineering and systematic linker optimization, the authors generate bacterial strains capable of selective and tunable gene expression in response to NIR stimulation. Overall, these results are an interesting expansion of the optogenetic toolkit into the NIR range. The cross-species functionality of the system, modularity, and orthogonality have the potential to make these tools useful for a range of applications.

Strengths:

(1) The authors introduce a novel class of near-infrared light-responsive two-component systems in bacteria, expanding the optogenetic toolbox into this spectral range.

(2) Through engineering and linker optimization, the authors achieve specific and tunable gene expression, with minimal cross-activation from red light in some cases.

(3) The authors show that the engineered systems function robustly in multiple bacterial strains, including laboratory E. coli, the probiotic E. coli Nissle 1917, and Agrobacterium tumefaciens.

(4) The combination of orthogonal two-component systems can allow for simultaneous and independent control of multiple gene expression pathways using different wavelengths of light.

(5) The authors explore the photophysical properties of the photosensors, investigating how environmental factors such as pH influence light sensitivity.

Comments on revisions:

The authors have addressed all my prior concerns.

Reviewer #3 (Public review):

Summary:

This paper by Meier et al introduces a new optogenetic module for regulation of bacterial gene expression based on "bathy-BphP" proteins. Their paper begins with a careful characterization of kinetics and pH dependence of a few family members, followed by extensive engineering to produce infrared-regulated transcriptional systems based on the authors' previous design of the pDusk and pDERusk systems, and closing with characterization of the systems in bacterial species relevant for biotechnology.

Strengths:

The paper is important from the perspective of fundamental protein characterization, since bathy-BphPs are relatively poorly characterized compared to their phytochrome and cyanobacteriochrome cousins. It is also important from a technology development perspective: the optogenetic toolbox currently lacks infrared-stimulated transcriptional systems. Infrared light offers two major advantages: it can be multiplexed with additional tools, and it can penetrate into deep tissues with ease relative to the more widely used blue light activated systems. The experiments are performed carefully and the manuscript is well written.

Weaknesses:

Some of the light-inducible responses described in this compelling paper are complex and difficult to rationalize, such as the dependence of light responses on linker length and differences in responses observed from the bathy-BphPs in isolation versus strains in which they are multiplexed. Nevertheless, the authors should be commended for carrying out rigorous experiments and reporting these results accurately. These are minor weaknesses in an overall very strong paper.

Author response:

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

Reviewer #1 (Public review):

Summary:

This is an interesting study characterizing and engineering so-called bathy phytochromes, i.e., those that respond to near infrared (NIR) light in the ground state, for optogenetic control of bacterial gene expression. Previously, the authors have developed a structure-guided approach to functionally link several light-responsive protein domains to the signaling domain of the histidine kinase FixL, which ultimately controls gene expression. Here, the authors use the same strategy to link bathy phytochrome light-responsive domains to FixL, resulting in sensors of NIR light. Interestingly, they also link these bathy phytochrome light-sensing domains to signaling domains from the tetrathionate-sensing SHK TtrS and the toluene-sensing SHK TodS, demonstrating the generality of their protein engineering approach more broadly across bacterial two-component systems.

This is an exciting result that should inspire future bacterial sensor design. They go on to leverage this result to develop what is, to my knowledge, the first system for orthogonally controlling the expression of two separate genes in the same cell with NIR and Red light, a valuable contribution to the field.

Finally, the authors reveal new details of the pH-dependent photocycle of bathy phytochromes and demonstrate that their sensors work in the gut - and plant-relevant strains E. coli Nissle 1917 and A. tumefaciens.

Strengths:

(1) The experiments are well-founded, well-executed, and rigorous.

(2) The manuscript is clearly written.

(3) The sensors developed exhibit large responses to light, making them valuable tools for ontogenetic applications.

(4) This study is a valuable contribution to photobiology and optogenetics.

We thank the reviewer for the positive verdict on our manuscript.

Weaknesses:

(1) As the authors note, the sensors are relatively insensitive to NIR light due to the rapid dark reversion process in bathy phytochromes. Though NIR light is generally non-phototoxic, one would expect this characteristic to be a limitation in some downstream applications where light intensities are not high (e.g., in vivo).

We principally concur with this reviewer’s assessment that delivery of light (of any color) into living tissue can be severely limited by absorption, reflection, and scattering. That notwithstanding, at least two considerations suggest that in-vivo deployment of the pNIRusk setups we presently advance may be feasible.

First, while the pNIRusk setups are indeed less light-sensitive compared to, e.g., our earlier redlight-responsive pREDusk and pDERusk setups (see Meier et al. Nat Commun 2024), we note that the overall light fluences required for triggering them are in the range of tens of µW per cm₂. By contrast, optogenetic experiments in vivo, in particular in the neurosciences, often employ light area intensities on the order of mW per cm₂ and above. Put another way, compared to the optogenetic tools used in these experiments, the pNIRusk setups are actually quite sensitive to light.

Second, sensitivity to NIR light brings the advantage of superior tissue penetration, see data reported by Weissleder Nat Biotech 2001 and Ash et al. Lasers Med Sci 2017 (both papers are cited in our manuscript). Based on these data, the intensity of blue light (450 nm) therefore falls off 5-10 times more strongly with penetration depth than that of NIR light (800 nm).

We have added a brief treatment of these aspects in the Discussion section.

(2) Though they can be multiplexed with Red light sensors, these bathy phytochrome NIR sensors are more difficult to multiplex with other commonly used light sensors (e.g., blue) due to the broad light responsivity of the Pfr state. This challenge may be overcome by careful dosing of blue light, as the authors discuss, but other bacterial NIR sensing systems with less cross-talk may be preferred in some applications.

The reviewer is correct in noting that, at least to a certain extent, the pNIRusk systems also respond to blue light owing to their Soret absorbance bands (see Fig. 1). That said, we note two points:

First, a given photoreceptor that preferentially responds to certain wavelengths, e.g., 700 nm in the case of conventional bacterial phytochromes (BphP), generally absorbs shorter wavelengths to some degree as well. Absorption of these shorter wavelengths suffices for driving electronic and/or vibronic transitions of the chromophore to higher energy levels which often give rise to productive photochemistry and downstream signal transduction. Put another way, a certain response of sensory photoreceptors to shorter wavelengths is hence fully expected and indeed experimentally borne out, as for instance shown by Ochoa-Fernandez et al. in the so-called PULSE setup (Nat Meth 2020, doi: 10.1038/s41592-020-0868-y).

Second, known BphPs share similar Pr and Pfr absorbance spectra. We therefore expect other BphP-based optogenetic setups to also respond to blue light to some degree. Currently, there are insufficient data to gauge whether individual BphPs systematically differ in their relative sensitivity to blue compared to red or NIR light. Arguably, pertinent experiments may be an interesting subject for future study.

Reviewer #2 (Public review):

Summary:

In this manuscript, Meier et al. engineer a new class of light-regulated two-component systems. These systems are built using bathy-bacteriophytochromes that respond to near-infrared (NIR) light. Through a combination of genetic engineering and systematic linker optimization, the authors generate bacterial strains capable of selective and tunable gene expression in response to NIR stimulation. Overall, these results are an interesting expansion of the optogenetic toolkit into the NIR range. The cross-species functionality of the system, modularity, and orthogonality have the potential to make these tools useful for a range of applications.

Strengths:

(1) The authors introduce a novel class of near-infrared light-responsive two-component systems in bacteria, expanding the optogenetic toolbox into this spectral range.

(2) Through engineering and linker optimization, the authors achieve specific and tunable gene expression, with minimal cross-activation from red light in some cases.

(3) The authors show that the engineered systems function robustly in multiple bacterial strains, including laboratory E. coli, the probiotic E. coli Nissle 1917, and Agrobacterium tumefaciens.

(4) The combination of orthogonal two-component systems can allow for simultaneous and independent control of multiple gene expression pathways using different wavelengths of light.

(5) The authors explore the photophysical properties of the photosensors, investigating how environmental factors such as pH influence light sensitivity.

Weaknesses:

(1) The expression of multi-gene operons and fluorescent reporters could impose a metabolic burden. The authors should present data comparing optical density for growth curves of engineered strains versus the corresponding empty-vector control to provide insight into the burden and overall impact of the system on host viability and growth.

In response to this comment, we have recorded growth kinetics of bacteria harboring the pNIRusk-DsRed plasmids or empty vectors under both inducing (i.e., under NIR light) and noninducing conditions (i.e., darkness). We did not observe systematic differences in the growth kinetics between the different cultures, thus suggesting that under the conditions tested there is no adverse effect on cell viability.

We include the new data in Suppl. Fig. 5c-d and refer to them in the main text.

(2) The manuscript consistently presents normalized fluorescence values, but the method of normalization is not clear (Figure 2 caption describes normalizing to the maximal fluorescence, but the maximum fluorescence of what?). The authors should provide a more detailed explanation of how the raw fluorescence data were processed. In addition, or potentially in exchange for the current presentation, the authors should include the raw fluorescence values in supplementary materials to help readers assess the actual magnitude of the reported responses.

We appreciate this valid comment and have altered the representation of the fluorescence data. All values for a given fluorescent protein (i.e., either DsRed or YPet) across all systems are now normalized to a single reference value, thus enabling direct comparison between experiments.

(3) Related to the prior point, it would be useful to have a positive control for fluorescence that could be used to compare results across different figure panels.

As all data are now normalized to the same reference value, direct comparison across all figures is enabled.

(4) Real-time gene expression data are not presented in the current manuscript, but it would be helpful to include a time-course for some of the key designs to help readers assess the speed of response to NIR light.

In response to this comment, we include in the revised manuscript induction kinetics of bacterial cultures bearing pNIRusk upon transfer to inducing NIR-light conditions. To this end, aliquots were taken at discrete timepoints, transcriptionally and translationally arrested, and analyzed for optical density and DsRed reporter fluorescence after allowing for chromophore maturation.

We include the new data in Suppl. Fig. 5e and refer to them in the manuscript.

Moreover, we note that the experiments in Agrobacterium tumefaciens used a luciferase reporter thus enabling the continuous monitoring of the light-induced expression kinetics. These data (unchanged in revision) are to be found in Suppl. Fig. 9.

Reviewer #3 (Public review):

Summary:

This paper by Meier et al introduces a new optogenetic module for the regulation of bacterial gene expression based on "bathy-BphP" proteins. Their paper begins with a careful characterization of kinetics and pH dependence of a few family members, followed by extensive engineering to produce infrared-regulated transcriptional systems based on the authors' previous design of the pDusk and pDERusk systems, and closing with characterization of the systems in bacterial species relevant for biotechnology.

Strengths:

The paper is important from the perspective of fundamental protein characterization, since bathyBphPs are relatively poorly characterized compared to their phytochrome and cyanobacteriochrome cousins. It is also important from a technology development perspective: the optogenetic toolbox currently lacks infrared-stimulated transcriptional systems. Infrared light offers two major advantages: it can be multiplexed with additional tools, and it can penetrate into deep tissues with ease relative to the more widely used blue light-activated systems. The experiments are performed carefully, and the manuscript is well written.

Weaknesses:

My major criticism is that some information is difficult to obtain, and some data is presented with limited interpretation, making it difficult to obtain intuition for why certain responses are observed. For example, the changes in red/infrared responses across different figures and cellular contexts are reported but not rationalized. Extensive experiments with variable linker sequences were performed, but the rationale for linker choices was not clearly explained. These are minor weaknesses in an overall very strong paper.

We are grateful for the positive take on our manuscript.

Reviewer #1 (Recommendations for the authors):

(1) As eLife is a broad audience journal, please define the Soret and Q-bands (line 125).

We concur and have added labels in fig. 1a that designate the Soret and Q bands.

(2) The initial (0) Ac design in Figure 2b is activated by NIR and Red light, albeit modestly. The authors state that this construct shows "constant reporter fluorescence, largely independent of illumination" (line 167). This language should be changed to reflect the fact that this Ac construct responds to both of these wavelengths.

Agreed. We have amended the text accordingly.

(3) pNIRusk Ac 0 appears to show a greater light response than pNIRusk Av -5. However, the authors claim that the former is not light-responsive and the latter is. This conclusion should be explained or changed.

The assignment of pNIRusk Av-5 as light-responsive is based on the relative difference in reporter fluorescence between darkness and illumination with either red or NIR light. Although the overall fluorescence is much lower in Av-5 than for Av-0, the relative change upon illumination is much more pronounced. We add a statement to this effect to the text.

(4) The authors state that "when combining DmDERusk-Str-YPet with AvTod+21-DsRed expression rose under red and NIR light, respectively, whereas the joint application of both light colors induced both reporter genes" (lines 258-261). In contrast, Figure 3c shows that application of both wavelengths of light results in exclusive activation of YPet expression. It appears the description of the data is wrong and must be corrected. That said, this error does not impact their conclusion that two separate target genes can be independently activated by NIR and red light.

We thank the reviewer for catching this error which we have corrected in the revised manuscript.

(5) Line 278: I don't agree with the authors' blanket statement that the use of upconversion nanoparticles is a "grave" limitation for NIR-light mediated activation of bacterial gene expression in vivo. The authors should either expound on the severity of the limitation or use more moderate language.

We have replaced the word ‘grave’ by ‘potential’ and thereby toned down our wording.

Reviewer #2 (Recommendations for the authors):

(1) Please include a discussion on the expected depth penetration of different light wavelengths. This is most relevant in the context of the discussion about how these NIR systems could be used with living therapeutics.

Given the heterogeneity of biological tissue, it is challenging to state precise penetration depths for different wavelengths of light. That said, blue light for instance is typically attenuated by biological tissue around 5 to 10 times as strongly as near-infrared light is.

We have expanded the Discussion chapter to cover these aspects.

(2) It would be helpful for Figure 2C (or supplementary) to also include the response to blue light stimulation.

We agree and have acquired pertinent data for the blue-light response. The new data are included in an updated Fig. 2c. Data acquired at varying NIR-light intensities, originally included in Fig. 2c, have been moved to Suppl. Fig. 5a-b.

(3) In Figure 4A, data on the response of E. coli Nissle to blue and red light are missing. Including this would help identify whether the reduced sensitivity to non-NIR wavelengths observed in the E. coli lab strain is preserved in the probiotic background.

In response to this comment, we have acquired pertinent data on E. coli Nissle. While the results were overall similar to those in the laboratory strain, the response to blue and NIR light was yet lower in the Nissle bacteria which stands to benefit optogenetic applications.

We have updated Fig. 4a accordingly. For clarity, we only show the data for AvNIRusk in the main paper but have relegated the data on AcNIRusk to Suppl. Fig. 8. (Note that this has necessitated a renumbering of the subsequent Suppl. Figs.)

(4) On many of the figures, there are thin gray lines that appear between the panels that it would be nice to eliminate because, in some cases, they cut through words and numbers.

The grey lines likely arose from embedding the figures into the text document. In the typeset manuscript, which has become available on the eLife webpage in the meantime, there are no such lines. That said, we will carefully check throughout the submission/publishing/proofing process lest these lines reappear.

(5) Page 7, line 155: "As not least seen" typo or awkward phrasing.

We have restructured the sentence and thereby hopefully clarified the unclear phrasing.

(6) Page 7, line 167: It does not appear to be the case that the initial pNIRusk designs show constant fluorescence that is largely independent of illumination. AcNIRusk shows an almost twofold change from dark to NIR. Reword this to avoid confusion.

We concur with this comment, similar to reviewer #1’s remark, and have adjusted the text accordingly.

(7) Page 8, line 174: Related to the previous point, AvNIRusk has one design that is very minimally light switchable (-5), so stating that six light switchable designs have been identified is also confusing.

As stated in our response to reviewer #1 above, the assignment of AvNIRusk-5 as light-switchable is based on the relative fluorescence change upon illumination. We have added an explanation to the text.

(8) Page 10, line 228-229: I was not able to find the data showing that expression levels were higher for the DmTtr systems than the pREDusk and pNIRusk setups. This may be an issue related to the normalization point. It was not clear to me how to compare these values.

We apologize for the initially unclear representation of the data. In response to this reviewer’s general comments above, we have now normalized all fluorescence values to a single reference value, thus allowing their direct comparison.

(9) Page 12, line 264: "finer-grained expression control can be exerted..." Either show data or adjust the language so that it is clear this is a prediction.

True, we have replaced ‘can’ by ‘could’.

(10) Page 25, line 590: CmpX13 cells have a reference that is given later, but it should be added where it first appears.

Agreed, we have added the reference in the indicated place.

(11) Page 25, line 592: define LB/Kan.

We had already defined this abbreviation further up but, for clarity, we have added it again in the indicated position.

(12) Page 40, line 946: "normalized by" rather than "to".

We have implemented the requested change in the indicated and several other positions of the manuscript.

(13) Figures 2C, 3C, and similar plots in the supplementary material would benefit from having a legend for the colors.

We agree and have added pertinent legends to the corresponding main and supplementary figures.

(14) As a reader, I had some trouble following all the acronyms. This is at the author's discretion, but I would eliminate ones that are not strictly essential (e.g. MTP for microtiter plate; I was unable to identify what "MCS" meant; look for other opportunities to remove acronyms).

In the revised manuscript, we have defined the abbreviation ‘MCS’ (for ‘multiple-cloning site’) upon first occurrence. We have decided to retain the abbreviation ‘MTP’ in the text.

(15) Could the authors briefly speculate on why A. tumefaciens activation with red light might occur?

While we can but speculate as to the underlying reasons for the divergent red-light response in A. tumefaciens, we discuss possible scenarios below.

Commonly, two-component systems (TCS) exhibit highly cooperative and steep responses to signal. As a consequence, even small differences in the intracellular amounts of phosphorylated and unphosphorylated response regulator (RR) can give to significantly changed gene-expression output. Put another way, the gene-expression output need not scale linearly with the extent of RR phosphorylation but, rather, is expected to show nonlinear dependence with pronounced thresholding effects.

Differences in the pertinent RR levels can for instance arise from variations in the expression levels of the pNIRusk system components between E. coli and A. tumefaciens. Moreover, the two bacteria greatly differ in their two-component-system (TCS) repertoire. Although TCSs are commonly well insulated from each other, cross-talk with endogenous TCSs, even if limited, may cause changes in the levels of phosphorylated RR and hence gene-expression output. In a similar vein, the RR can also be phosphorylated and dephosphorylated non-enzymatically, e.g., by reaction with high-energy anhydrides (such as acetyl phosphate) and hydrolysis, respectively. Other potential origins for the divergent red-light response include differences in the strength of the promoters driving expression of the pNIRusk system components and the fluorescent/luminescent reporters, respectively.

(16) It would be helpful for the authors to briefly explain why they needed to switch to luminescence from fluorescence for the A. tumeraciens studies.

While there was no strict necessity to switch from the fluorescence-based system used in E. coli to a luminescence-based system in A. tumefaciens, we opted for luminescence based on prior experience with other Alphaproteobacteria (e.g., 10.1128/mSystems.00893-21), where luminescence offered significant advantages. Specifically, it provides essentially background-free signal detection and greater sensitivity for monitoring gene expression. In addition, as demonstrated in Suppl. Fig. 9c and d, the luminescence system enables real-time tracking of gene expression dynamics, which further supported its use in our experimental setup (see our response to reviewer #2’s general comments).

(17) This is a very minor comment that the authors can take or leave, but I got hung up on the word "implement" when it appeared a few times in the manuscript because I tended to read it as "put a plan into place" rather than its other meaning.

In the abstract, we have replaced one instance of the word ‘implement’ by ‘instrument’.

(18) The authors should include the relevant constructs on AddGene or another public strainsharing service.

We whole-heartedly subscribe to the idea of freely sharing research materials with fellow scientists. Therefore, we had already deposited the most relevant AvNIRusk in Addgene, even prior to the initial submission of the manuscript (accession number 235084). In the meantime, we have released the deposition, and the plasmid can be obtained from Addgene since May 15_th of this year.

Reviewer #3 (Recommendations for the authors):

Suggestion for improvement:

This paper relies heavily on variations in linker sequences to shift responses. I am familiar with prior work from the Moglich lab in which helical linkers were employed to shift responses in synthetic two-component systems, with interesting periodicity in responses with every 7 residues (as expected for an alpha helix) and inversion of responses at smaller linker shifts. There is no mention in this paper whether their current engineering follows a similar rationale, what types of linkers are employed (e.g. flexible vs helical), and whether there is an interpretation for how linker lengths alter responses. Can you explain what classes of linker sequences are used throughout Figures 2 and 3, and whether length or periodicity affects the outcome? This would be very helpful for readers who are new to this approach, or if the rationale here differs from the authors' prior work.

The PATCHY approach employed at present followed a closely similar rationale as in our previous studies. That is, linkers were extended/shortened and varied in their sequence by recombining different fragments of the natural linkers of the parental receptors, i.e., the bacteriophytochrome and the FixL sensor histidine kinase, respectively. We have added a statement to this effect in the text and a reference to Suppl. Fig. 3 which illustrates the principal approach.

Compared to our earlier studies, we isolated fewer receptor variants supporting light-regulated responses, despite covering a larger sequence space. Owing to the sparsity of the light-regulated variants, an interpretation of the linker properties and their correlation with light-regulated activity is challenging. Although doubtless unsatisfying from a mechanistic viewpoint, we therefore refrain from a pertinent discussion which would be premature and speculative at this point. As the reviewer raises a valid and important point, we have expanded the text by referring to our earlier studies and the observed dependence of functional properties on linker composition.

It is sometimes difficult to intuit or rationalize the differences in red/IR sensitivity across closely related variants. An important example appears in Figure 3C vs 3B. I think the AvTod+21 in 3B should be the equivalent to the DsRed response in the second column of 3C (AvTod+21 + DmDERusk), except, of course, that the bacteria in 3C carry an additional plasmid for the DERusk system. However, in 3B, the response to red light is substantial - ~50% as strong as that for IR, whereas in 3C, red light elicits no response at all. What is the difference? The reason this is important is that the AvTod+21 and DMDERusk represent the best "orthogonal" red and infrared light responses, but this is not at all obvious from 3B, where AvTod+21 still causes a substantial (and for orthogonality, undesirable) response under red light. Perhaps subtle differences in expression level due to plasmid changes cause these differences in light responses? Could the authors test how the expression level affects these responses? The paper would be greatly improved if observations of the diverse red/IR responses could be rationalized by some design criteria.

As noted above in our response to reviewer #2, we have now normalized all fluorescence readings to joint reference values, thus allowing a better comparison across experiments.

The reviewer is correct in noting that upon multiplexing, the individual plasmid systems support lower fluorescence levels than when used in isolation. We speculate that the combination of two plasmids may affect their copy numbers (despite the use of different resistance markers and origins of replications) and hence their performance. Likewise, the cellular metabolism may be affected when multiple plasmids are combined. These aspects may well account for the absent red-light response in AvTod+21 in the multiplexing experiments which is – indeed – unexpected. As, at present, we cannot provide a clear rationalization for this effect, we recommend verifying the performance of the plasmid setups when multiplexing.

The paper uses "red" and "infrared" to refer to ~624 nm and ~800 nm light, respectively. I wonder whether it might be possible to shift these peak wavelengths to obtain even better separation for the multiplexing experiments. Perhaps shifting the specific red wavelength could result in better separation between DERusk and AvTod systems, for example? Could the authors comment on this (maybe based on action spectra of their previously developed tools) or perhaps test a few additional stimulation wavelengths?

The choice of illumination wavelengths used in these experiments is dictated by the LED setups available for illumination of microtiter plates. On the one hand, we are using an SMD (surface-mount device) three-color LED with a fixed wavelength of the red channel around 624 nm (see Hennemann et al., 2018). On the other hand, we are deploying a custom-built device with LEDs emitting at around 800 nm (see Stüven et al., 2019 and this work). Adjusting these wavelengths is therefore challenging, although without doubt potentially interesting.

To address this reviewer comment, we have added a statement to the text that the excitation wavelengths may be varied to improve multiplexed applications.

Additional minor comments:

(1) Figure 2C: It would be very helpful to place a legend on the figure panel for what the colors indicate, since they are unique to this panel and non-intuitive.

This comment coincides with one by reviewer #2, and we have added pertinent legends to this and related supplementary figures.

(2) Figure 3C: it is not obvious which system uses DsRed and which uses YPet in each combination, since the text indicates that all combinations were cloned, and this is not clearly described in the legend. Is it always the first construct in the figure legend listed for DsRed and the second for YPet?

For clarification, we have revised the x-axis labels in Fig. 3C. (And yes, it is as this reviewer surmises: the first of the two constructs harbored DsRed and the second one YPet.)

Hi Chris,

I think we should add a calendar option for selecting dates (similar to flight booking) instead of just writing out the months. The tentative dates are flexible, but they’ll likely be close to the actual days. Plus, this approach would give a more professional appearance. Please check with Nash and proceed if it makes sense.

Thanks.

eLife Assessment

This important manuscript presents a thorough analysis of the evolution of Major Histocompatibility Complex gene families across Primates. A key strength of this analysis is the use of state-of-the-art phylogenetic methods to estimate rates of gene gain and loss, accounting for the notorious difficulty to properly assemble MHC genomic regions. Overall the evidence for the authors' conclusions -- that there is considerable diversity in how MHC diversity is deployed across species -- are compelling.

Joint Public Review:

Summary:

The Major Histocompatibility Complex (MHC) region is a collection of numerous genes involved in both innate and adaptive immunity. MHC genes are famed for their role in rapid evolution and extensive polymorphism in a variety of vertebrates. This paper presents a summary of gene-level gain and loss of orthologs and paralogs within MHC across the diversity of primates, using publicly available data.

Strengths:

This paper provides a strong case that MHC genes are rapidly gained (by paralog duplication) and lost over millions of years of macroevolution. The authors are able to identify MHC loci by homology across species, and from this infer gene duplications and losses using phylogenetic analyses. There is a remarkable amount of genic turnover, summarized in Figure 6 and Figure 7, either of which might be a future textbook figure of immune gene family evolution. The authors draw on state-of-the-art phylogenetic methods, and their inferences are robust.

Editorial note:

The authors have responded to the previous reviews and the Assessment was updated without involving the reviewers again.

Author response:

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

Reviewer #1 (Public review):

Summary:

The Major Histocompatibility Complex (MHC) region is a collection of numerous genes involved in both innate and adaptive immunity. MHC genes are famed for their role in rapid evolution and extensive polymorphism in a variety of vertebrates. This paper presents a summary of gene-level gain and loss of orthologs and paralogs within MHC across the diversity of primates, using publicly available data.

Strengths:

This paper provides a strong case that MHC genes are rapidly gained (by paralog duplication) and lost over millions of years of macroevolution. The authors are able to identify MHC loci by homology across species, and from this infer gene duplications and losses using phylogenetic analyses. There is a remarkable amount of genic turnover, summarized in Figure 6 and Figure 7, either of which might be a future textbook figure of immune gene family evolution. The authors draw on state-of-the-art phylogenetic methods, and their inferences are robust insofar as the data might be complete enough to draw such conclusions.

Weaknesses:

One concern about the present work is that it relies on public databases to draw inferences about gene loss, which is potentially risky if the publicly available sequence data are incomplete. To say, for example, that a particular MHC gene copy is absent in a taxon (e.g., Class I locus F absent in Guenons according to Figure 1), we need to trust that its absence from the available databases is an accurate reflection of its absence in the genome of the actual organisms. This may be a safe assumption, but it rests on the completeness of genome assembly (and gene annotations?) or people uploading relevant data. This reviewer would have been far more comfortable had the authors engaged in some active spot-checking, doing the lab work to try to confirm absences at least for some loci and some species. Without this, a reader is left to wonder whether gene loss is simply reflecting imperfect databases, which then undercuts confidence in estimates of rates of gene loss.

Indeed, just because a locus has not been confirmed in a species does not necessarily mean that it is absent. As we explain in the Figure 1 caption, only a few species have had their genomes extensively studied (gray background), and only for these species does the absence of a point in this figure mean that a locus is absent. The white background rows represent species that are not extensively studied, and we point out that the absence of a point does not mean that a locus is absent from the species, rather undiscovered. We have also added a parenthetical to the text to explain this (line 156): “Only species with rows highlighted in gray have had their MHC regions extensively studied (and thus only for these rows is the absence of a gene symbol meaningful).”

While we agree that spot-checking may be a helpful next step, one of the goals of this manuscript is to collect and synthesize the enormous volume of MHC evolution research in the primates, which will serve as a jumping-off point for other researchers to perform important wet lab work.

Some context is useful for comparing rates of gene turnover in MHC, to other loci. Changing gene copy numbers, duplications, and loss of duplicates, are common it seems across many loci and many organisms; is MHC exceptional in this regard, or merely behaving like any moderately large gene family? I would very much have liked to see comparable analyses done for other gene families (immune, like TLRs, or non-immune), and quantitative comparisons of evolutionary rates between MHC versus other genes. Does MHC gene composition evolve any faster than a random gene family? At present readers may be tempted to infer this, but evidence is not provided.

Our companion paper (Fortier and Pritchard, 2025) demonstrates that the MHC is a unique locus in many regards, such as its evidence for deep balancing selection and its excess of disease associations. Thus, we expect that it is evolving faster than any random gene family. It would be interesting to repeat this analysis for other gene families, but that is outside of the scope of this project. Additionally, allele databases for other gene families are not nearly as developed, but as more alleles become available for other polymorphic families, a comparable analysis could become possible.

We have added a paragraph to the discussion (lines 530-546) to clarify that we do not know for certain whether the MHC gene family is evolving rapidly compared to other gene families.

While on the topic of making comparisons, the authors make a few statements about relative rates. For instance, lines 447-8 compare gene topology of classical versus non-classical genes; and line 450 states that classical genes experience more turnover. But there are no quantitative values given to these rates to provide numerical comparisons, nor confidence intervals provided (these are needed, given that they are estimates), nor formal statistical comparisons to confirm our confidence that rates differ between types of genes.

More broadly, the paper uses sophisticated phylogenetic methods, but without taking advantage of macroevolutionary comparative methods that allow model-based estimation of macroevolutionary rates. I found the lack of quantitative measurements of rates of gene gain/loss to be a weakness of the present version of the paper, and something that should be readily remedied. When claiming that MHC Class I genes "turn over rapidly" (line 476) - what does rapidly mean? How rapidly? How does that compare to rates of genetic turnover at other families? Quantitative statements should be supported by quantitative estimates (and their confidence intervals).

These statements refer to qualitative observations, so we cannot provide numerical values. We simply conclude that certain gene groups evolve faster or slower based on the species and genes present in each clade. It is difficult to provide estimates because of the incomplete sampling of genes that survived to the present day. In addition, the presence or absence of various orthologs in different species still needs to be confirmed, at which point it might be useful to be more quantitative. We have also added a paragraph to the discussion to address this concern and advocate for similar analyses of other gene families in the future when more data is available (lines 530-546).

The authors refer to 'shared function of the MHC across species' (e.g. line 22); while this is likely true, they are not here presenting any functional data to confirm this, nor can they rule out neofunctionalization or subfunctionalization of gene duplicates. There is evidence in other vertebrates (e.g., cod) of MHC evolving appreciably altered functions, so one may not safely assume the function of a locus is static over long macroevolutionary periods, although that would be a plausible assumption at first glance.

Indeed, we cannot assume that the function of a locus is static across time, especially for the MHC region. In our research, we read hundreds of papers that each focused on a small number of species or genes and gathered some information about them, sometimes based on functional experiments and sometimes on measures such as dN/dS. These provide some indication of a gene’s broad classification in a species or clade, even if the evidence is preliminary. Where possible, we used this preliminary evidence to give genes descriptors “classical,” “non-classical,” “dual characteristics,” “pseudogene,” “fixed”, or “unfixed.” Sometimes multiple individuals and haplotypes were analyzed, so we could even assign a minimum number of gene copies present in a species. We have aggregated all of these references into Supplementary Table 1 (for Class I/Figure 1) and Supplementary Table 2 (for Class II/Figure 2) along with specific details about which data points in these figures that each reference supports. We realize that many of these classifications are based on a small number of individuals or indirect measures, so they may change in the future as more functional data is generated.

Reviewer #2 (Public review):

Summary:

The authors aim to provide a comprehensive understanding of the evolutionary history of the Major Histocompatibility Complex (MHC) gene family across primate species. Specifically, they sought to:

(1) Analyze the evolutionary patterns of MHC genes and pseudogenes across the entire primate order, spanning 60 million years of evolution.

(2) Build gene and allele trees to compare the evolutionary rates of MHC Class I and Class II genes, with a focus on identifying which genes have evolved rapidly and which have remained stable.

(3) Investigate the role of often-overlooked pseudogenes in reconstructing evolutionary events, especially within the Class I region.

(4) Highlight how different primate species use varied MHC genes, haplotypes, and genetic variation to mount successful immune responses, despite the shared function of the MHC across species.

(5) Fill gaps in the current understanding of MHC evolution by taking a broader, multi-species perspective using (a) phylogenomic analytical computing methods such as Beast2, Geneconv, BLAST, and the much larger computing capacities that have been developed and made available to researchers over the past few decades, (b) literature review for gene content and arrangement, and genomic rearrangements via haplotype comparisons.

(6) The authors overall conclusions based on their analyses and results are that 'different species employ different genes, haplotypes, and patterns of variation to achieve a successful immune response'.

Strengths:

Essentially, much of the information presented in this paper is already well-known in the MHC field of genomic and genetic research, with few new conclusions and with insufficient respect to past studies. Nevertheless, while MHC evolution is a well-studied area, this paper potentially adds some originality through its comprehensive, cross-species evolutionary analysis of primates, focus on pseudogenes and the modern, large-scale methods employed. Its originality lies in its broad evolutionary scope of the primate order among mammals with solid methodological and phylogenetic analyses.

The main strengths of this study are the use of large publicly available databases for primate MHC sequences, the intensive computing involved, the phylogenetic tool Beast2 to create multigene Bayesian phylogenetic trees using sequences from all genes and species, separated into Class I and Class II groups to provide a backbone of broad relationships to investigate subtrees, and the presentation of various subtrees as species and gene trees in an attempt to elucidate the unique gene duplications within the different species. The study provides some additional insights with summaries of MHC reference genomes and haplotypes in the context of a literature review to identify the gene content and haplotypes known to be present in different primate species. The phylogenetic overlays or ideograms (Figures 6 and 7) in part show the complexity of the evolution and organisation of the primate MHC genes via the orthologous and paralogous gene and species pathways progressively from the poorly-studied NWM, across a few moderately studied ape species, to the better-studied human MHC genes and haplotypes.

Weaknesses:

The title 'The Primate Major Histocompatibility Complex: An Illustrative Example of GeneFamily Evolution' suggests that the paper will explore how the Major Histocompatibility Complex (MHC) in primates serves as a model for understanding gene family evolution. The term 'Illustrative Example' in the title would be appropriate if the paper aimed to use the primate Major Histocompatibility Complex (MHC) as a clear and representative case to demonstrate broader principles of gene family evolution. That is, the MHC gene family is not just one instance of gene family evolution but serves as a well-studied, insightful example that can highlight key mechanisms and concepts applicable to other gene families. However, this is not the case, this paper only covers specific details of primate MHC evolution without drawing broader lessons to any other gene families. So, the term 'Illustrative Example' is too broad or generalizing. In this case, a term like 'Case Study' or simply 'Example' would be more suitable. Perhaps, 'An Example of Gene Family Diversity' would be more precise. Also, an explanation or 'reminder' is suggested that this study is not about the origins of the MHC genes from the earliest jawed vertebrates per se (~600 mya), but it is an extension within a subspecies set that has emerged relatively late (~60 mya) in the evolutionary divergent pathways of the MHC genes, systems, and various vertebrate species.

Thank you for your input on the title; we have changed it to “A case study of gene family evolution” instead.

Thank you also for pointing out the potential confusion about the time span of our study. We have added “Having originated in the jawed vertebrates,” to a sentence in the introduction (lines 38-39). We have also added the sentence “Here, we focus on the primates, spanning approximately 60 million years within the over 500-million-year evolution of the family \citep{Flajnik2010}.“ to be more explicit about the context for our work (lines 59-61).

Phylogenomics. Particular weaknesses in this study are the limitations and problems associated with providing phylogenetic gene and species trees to try and solve the complex issue of the molecular mechanisms involved with imperfect gene duplications, losses, and rearrangements in a complex genomic region such as the MHC that is involved in various effects on the response and regulation of the immune system. A particular deficiency is drawing conclusions based on a single exon of the genes. Different exons present different trees. Which are the more reliable? Why were introns not included in the analyses? The authors attempt to overcome these limitations by including genomic haplotype analysis, duplication models, and the supporting or contradictory information available in previous publications. They succeed in part with this multidiscipline approach, but much is missed because of biased literature selection. The authors should include a paragraph about the benefits and limitations of the software that they have chosen for their analysis, and perhaps suggest some alternative tools that they might have tried comparatively. How were problems with Bayesian phylogeny such as computational intensity, choosing probabilities, choosing particular exons for analysis, assumptions of evolutionary models, rates of evolution, systemic bias, and absence of structural and functional information addressed and controlled for in this study?

We agree that different exons have different trees, which is exactly why we repeated our analysis for each exon in order to compare and contrast them. In particular, the exons encoding the binding site of the resulting protein (exons 2 and 3 for Class I and exon 2 for Class II) show evidence for trans-species polymorphism and gene conversion. These phenomena lead to trees that do not follow the species tree and are fascinating in and of themselves, which we explore in detail in our companion paper (Fortier and Pritchard, 2025). Meanwhile, the non-peptide-binding extracellular-domain-encoding exon (exon 4 for Class I and exon 3 for Class II) is comparably sized to the binding-site-encoding exons and provides an interesting functional contrast. As this exon is likely less affected by trans-species polymorphism, gene conversion, and convergent evolution, we present results from it most often in the main text, though we occasionally touch on differences between the exons. See lines 191-196, 223-226, and 407-414 for some examples of how we discuss the exons in the text. Additionally, all trees from all of these exons can be found in the supplement. 

We agree that introns would valuable to study in this context. Even though the non--binding-site-encoding exons are probably *less* affected by trans-species polymorphism, gene conversion, and convergent evolution, they are still functional. The introns, however, experience much more relaxed selection, if any, and comparing their trees to those for the exons would be valuable and illuminating. We did not generate intron trees for two reasons. Most importantly, there is a dearth of data available for the introns; in the databases we used, there was often intron data available only for human, chimpanzee, and sometimes macaque, and only for a small subset of the genes. This limitation is at odds with the comprehensive, many-gene-many-species approach which we feel is the main novelty of this work. Secondly, the introns that *are* available are difficult to align. Even aligning the exons across such a highly-diverged set of genes and pseudogenes was difficult and required manual effort. The introns proved even more difficult to try to align across genes. In the future, when more intron data is available and sufficient effort is put into aligning them, it will be possible and desirable to do a comparable analysis. We also added a sentence to the “Data” section to briefly explain why we did not include introns (lines 134-135).

We explain our Bayesian phylogenetics approach in detail in the Methods (lines 650-725), including our assumptions and our solutions to challenges specific to this application. For further explanation of the method itself, we suggest reading the original BEAST and BEAST2 papers (Drummond & Rambaut (2007), Drummond et al. (2012), Bouckaert et al. (2014), and Bouckaert et al. (2019)). Known structural and functional information helped us validate the alignments we used in this study, but the fact that such information is not fully known for every gene and species should not affect the method itself.

Gene families as haplotypes. In the Introduction, the MHC is referred to as a 'gene family', and in paragraph 2, it is described as being united by the 'MHC fold', despite exhibiting 'very diverse functions'. However, the MHC region is more accurately described as a multigene region containing diverse, haplotype-specific Conserved Polymorphic Sequences, many of which are likely to be regulatory rather than protein-coding. These regulatory elements are essential for controlling the expression of multiple MHC-related products, such as TNF and complement proteins, a relationship demonstrated over 30 years ago. Non-MHC fold loci such as TNF, complement, POU5F1, lncRNA, TRIM genes, LTA, LTB, NFkBIL1, etc, are present across all MHC haplotypes and play significant roles in regulation. Evolutionary selection must act on genotypes, considering both paternal and maternal haplotypes, rather than on individual genes alone. While it is valuable to compile databases for public use, their utility is diminished if they perpetuate outdated theories like the 'birth-and-death model'. The inclusion of prior information or assumptions used in a statistical or computational model, typically in Bayesian analysis, is commendable, but they should be based on genotypic data rather than older models. A more robust approach would consider the imperfect duplication of segments, the history of their conservation, and the functional differences in inheritance patterns. Additionally, the MHC should be examined as a genomic region, with ancestral haplotypes and sequence changes or rearrangements serving as key indicators of human evolution after the 'Out of Africa' migration, and with disease susceptibility providing a measurable outcome. There are more than 7000 different HLA-B and -C alleles at each locus, which suggests that there are many thousands of human HLA haplotypes to study. In this regard, the studies by Dawkins et al (1999 Immunol Rev 167,275), Shiina et al. (2006 Genetics 173,1555) on human MHC gene diversity and disease hitchhiking (haplotypes), and Sznarkowska et al. (2020 Cancers 12,1155) on the complex regulatory networks governing MHC expression, both in terms of immune transcription factor binding sites and regulatory non-coding RNAs, should be examined in greater detail, particularly in the context of MHC gene allelic diversity and locus organization in humans and other primates.

Thank you for these comments. To clarify that the MHC “region” is different from (and contains) the MHC “gene family” as we describe it, we changed a sentence in the abstract (lines 8-10) from “One large gene family that has experienced rapid evolution is the Major Histocompatibility Complex (MHC), whose proteins serve critical roles in innate and adaptive immunity.” to “One large gene family that has experienced rapid evolution lies within the Major Histocompatibility Complex (MHC), whose proteins serve critical roles in innate and adaptive immunity.” We know that the region is complex and contains many other genes and regulatory sequences; Figure 1 of our companion paper (Fortier and Pritchard, 2025) depicts these in order to show the reader that the MHC genes we focus on are just one part of the entire region.

We love the suggestion to look at the many thousands of alleles present at each of the classical loci. This is the focus of our complimentary paper (Fortier and Pritchard, 2025) which explores variation at the allele level. In the current paper, we look mainly at the differences between genes and the use of different genes in different species.

Diversifying and/or concerted evolution. Both this and past studies highlight diversifying selection or balancing selection model is the dominant force in MHC evolution. This is primarily because the extreme polymorphism observed in MHC genes is advantageous for populations in terms of pathogen defence. Diversification increases the range of peptides that can be presented to T cells, enhancing the immune response. The peptide-binding regions of MHC genes are highly variable, and this variability is maintained through selection for immune function, especially in the face of rapidly evolving pathogens. In contrast, concerted evolution, which typically involves the homogenization of gene duplicates through processes like gene conversion or unequal crossing-over, seems to play a minimal role in MHC evolution. Although gene duplication events have occurred in the MHC region leading to the expansion of gene families, the resulting paralogs often undergo divergent evolution rather than being kept similar or homozygous by concerted evolution. Therefore, unlike gene families such as ribosomal RNA genes or histone genes, where concerted evolution leads to highly similar copies, MHC genes display much higher levels of allelic and functional diversification. Each MHC gene copy tends to evolve independently after duplication, acquiring unique polymorphisms that enhance the repertoire of antigen presentation, rather than undergoing homogenization through gene conversion. Also, in some populations with high polymorphism or genetic drift, allele frequencies may become similar over time without the influence of gene conversion. This similarity can be mistaken for gene conversion when it is simply due to neutral evolution or drift, particularly in small populations or bottlenecked species. Moreover, gene conversion might contribute to greater diversity by creating hybrids or mosaics between different MHC genes. In this regard, can the authors indicate what percentage of the gene numbers in their study have been homogenised by gene conversion compared to those that have been diversified by gene conversion?

We appreciate the summary, and we feel we have appropriately discussed both gene conversion and diversifying selection in the context of the MHC genes. Because we cannot know for sure when and where gene conversion has occurred, we cannot quantify percentages of genes that have been homogenized or diversified.  

Duplication models. The phylogenetic overlays or ideograms (Figures 6 and 7) show considerable imperfect multigene duplications, losses, and rearrangements, but the paper's Discussion provides no in-depth consideration of the various multigenic models or mechanisms that can be used to explain the occurrence of such events. How do their duplication models compare to those proposed by others? For example, their text simply says on line 292, 'the proposed series of events is not always consistent with phylogenetic data'. How, why, when? Duplication models for the generation and extension of the human MHC class I genes as duplicons (extended gene or segmental genomic structures) by parsimonious imperfect tandem duplications with deletions and rearrangements in the alpha, beta, and kappa blocks were already formulated in the late 1990s and extended to the rhesus macaque in 2004 based on genomic haplotypic sequences. These studies were based on genomic sequences (genes, pseudogenes, retroelements), dot plot matrix comparisons, and phylogenetic analyses of gene and retroelement sequences using computer programs. It already was noted or proposed in these earlier 1999 studies that (1) the ancestor of HLA-P(90)/-T(16)/W(80) represented an old lineage separate from the other HLA class I genes in the alpha block, (2) HLA-U(21) is a duplicated fragment of HLA-A, (3) HLA-F and HLA-V(75) are among the earliest (progenitor) genes or outgroups within the alpha block, (4) distinct Alu and L1 retroelement sequences adjoining HLA-L(30), and HLA-N genomic segments (duplicons) in the kappa block are closely related to those in the HLA-B and HLA-C in the beta block; suggesting an inverted duplication and transposition of the HLA genes and retroelements between the beta and kappa regions. None of these prior human studies were referenced by Fortier and Pritchard in their paper. How does their human MHC class I gene duplication model (Fig. 6) such as gene duplication numbers and turnovers differ from those previously proposed and described by Kulski et al (1997 JME 45,599), (1999 JME 49,84), (2000 JME 50,510), Dawkins et al (1999 Immunol Rev 167,275), and Gaudieri et al (1999 GR 9,541)? Is this a case of reinventing the wheel?

Figures 6 and 7 are intended to synthesize and reconcile past findings and our own trees, so they do not strictly adhere to the findings of any particular study and cannot fully match all studies. In the supplement, Figure 6 - figure supplement 1 and Figure 7 - figure supplement 1 duly credit all of the past work that went into making these trees. Most previous papers focus on just one aspect of these trees, such as haplotypes within a species, a specific gene or allelic lineage relationship, or the branching pattern of particular gene groups. We believe it was necessary to bring all of these pieces of evidence together. Even among papers with the same focus (to understand the block duplications that generated the current physical layout of the MHC), results differ. For example, Geraghty (1992), Hughes (1995), Kulski (2004)/Kulski (2005),  and Shiina (1999) all disagree on the exact branching order of the genes MHC-W, -P, and -T, and of MHC-G, -J, and -K. While the Kulski studies you pointed out were very thorough for their era, they still only relied on data from three species and one haplotype per species. Our work is not intended to replace or discredit these past works, simply build upon them with a larger set of species and sequences. We hope the hypotheses we propose in Figures 6 and 7 can help unify existing research and provide a more easily accessible jumping-off-point for future work.

Results. The results are presented as new findings, whereas most if not all of the results' significance and importance already have been discussed in various other publications. Therefore, the authors might do better to combine the results and discussion into a single section with appropriate citations to previously published findings presented among their results for comparison. Do the trees and subsets differ from previous publications, albeit that they might have fewer comparative examples and samples than the present preprint? Alternatively, the results and discussion could be combined and presented as a review of the field, which would make more sense and be more honest than the current format of essentially rehashing old data.

In starting this project, we found that a large barrier to entry to this field of study is the immense amount of published literature over 30+ years. It is both time-consuming and confusing to read up on the many nuances of the MHC genes, their changing names, and their evolution, making it difficult to start new, innovative projects. We acknowledge that while our results are not entirely novel, the main advantage of our work is that it provides a thorough, comprehensive starting point for others to learn about the MHC quickly and dive into new research. We feel that we have appropriately cited past literature in both the main text, appendices, and supplement, so that readers may dive into a particular area with ease.

Minor corrections:

(1) Abstract, line 19: 'modern methods'. Too general. What modern methods?

To keep the abstract brief, the methods are introduced in the main text when each becomes relevant as well as in the methods section.

(2) Abstract, line 25: 'look into [primate] MHC evolution.' The analysis is on the primate MHC genes, not on the entire vertebrate MHC evolution with a gene collection from sharks to humans. The non-primate MHC genes are often differently organised and structurally evolved in comparison to primate MHC.

Thank you! We have added the word “primate” to the abstract (line 25).

(3) Introduction, line 113. 'In a companion paper (Fortier and Pritchard, 2024)' This paper appears to be unpublished. If it's unpublished, it should not be referenced.

This paper is undergoing the eLife editorial process at the same time; it will have a proper citation in the final version.

(4) Figures 1 and 2. Use the term 'gene symbols' (circle, square, triangle, inverted triangle, diamond) or 'gene markers' instead of 'points'. 'Asterisks "within symbols" indicate new information.

Thank you, the word “symbol” is much clearer! We have changed “points” to “symbols” in the captions for Figure 1, Figure 1 - figure supplement 1, Figure 2, and Figure 2 - figure supplement 1. We also changed this in the text (lines 157-158 and 170).

(5) Figures. A variety of colours have been applied for visualisation. However, some coloured texts are so light in colour that they are difficult to read against a white background. Could darker colours or black be used for all or most texts?

With such a large number of genes and species to handle in this work, it was nearly impossible to choose a set of colors that were distinct enough from each other. We decided to prioritize consistency (across this paper, its supplement, and our companion paper) as well as at-a-glance grouping of similar sequences. Unfortunately, this means we had to sacrifice readability on a white background, but readers may turn to the supplement if they need to access specific sequence names.

(6) Results, line 135. '(Fortier and Pritchard, 2024)' This paper appears to be unpublished. If it's unpublished, it should not be referenced.

Repeat of (3). This paper is undergoing the eLife editorial process at the same time; it will have a proper citation in the final version.

(7) Results, lines 152 to 153, 164, 165, etc. 'Points with an asterisk'. Use the term 'gene symbols' (circle, square, triangle, inverted triangle, diamond) or 'gene markers' instead of 'points'. A point is a small dot such as those used in data points for plotting graphs .... The figures are so small that the asterisks in the circles, squares, triangles, etc, look like points (dots) and the points/asterisks terminology that is used is very confusing visually.

Repeat of (4). Thank you, the word “symbol” is much clearer! We have changed “points” to “symbols” in the captions for Figure 1, Figure 1 - figure supplement 1, Figure 2, and Figure 2 - figure supplement 1. We also changed this in the text (lines 157-158 and 170).

(8) Line 178 (BEA, 2024) is not listed alphabetically in the References.

Thank you for catching this! This reference maps to the first bibliography entry, “SUMMARIZING POSTERIOR TREES.” We are unsure how to cite a webpage that has no explicit author within the eLife Overleaf template, so we will consult with the editor.

(9) Lines 188-190. 'NWM MHC-G does not group with ape/OWM MHC-G, instead falling outside of the clade containing ape/OWM MHC-A, -G, -J and -K.' This is not surprising given that MHC-A, -G, -J, and -K are paralogs of each other and that some of them, especially in NWM have diverged over time from the paralogs and/or orthologs and might be closer to one paralog than another and not be an actual ortholog of OWM, apes or humans.

We included this sentence to clarify the relationships between genes and to help describe what is happening in Figure 6. Figure 6 - figure supplement 1 includes all of the references that go into such a statement and Appendix 3 details our reasoning for this and other statements.

(10) Line 249. Gene conversion: This is recombination between two different genes where a portion of the genes are exchanged with one another so that different portions of the gene can group within one or other of the two gene clades. Alternatively, the gene has been annotated incorrectly if the gene does not group within either of the two alternative clades. Another possibility is that one or two nucleotide mutations have occurred without a recombination resulting in a mistaken interpretation or conclusion of a recombination event. What measures are taken to avoid false-positive conclusions? How many MHC gene conversion (recombination) events have occurred according to the authors' estimates? What measures are taken to avoid false-positive conclusions?

All of these possibilities are certainly valid. We used the program GENECONV to infer gene conversion events, but there is considerable uncertainty owing to the ages of the genes and the inevitable point mutations that have occurred post-event. Gene conversion was not the focus of our paper, so we did our best to acknowledge it (and the resulting differences between trees from different exons) without spending too much time diving into it. A list of inferred gene conversion events can be found in Figure 3 - source data 1 and Figure 4 - source data 1.

(11) Lines 284-286. 'The Class I MHC region is further divided into three polymorphic blocks-alpha, beta, and kappa blocks-that each contains MHC genes but are separated by well-conserved non-MHC genes.' The MHC class I region was first designated into conserved polymorphic duplication blocks, alpha and beta by Dawkins et al (1999 Immunol Rev 167,275), and kappa by Kulski et al (2002 Immunol Rev 190,95), and should be acknowledged (cited) accordingly.

Thank you for catching this! We have added these citations (lines 302-303)!

(12) Lines 285-286. 'The majority of the Class I genes are located in the alpha-block, which in humans includes 12 MHC genes and pseudogenes.' This is not strictly correct for many other species, because the majority of class I genes might be in the beta block of new and old-world monkeys, and the authors haven't provided respective counts of duplication numbers to show otherwise. The alpha block in some non-primate mammalian species such as pigs, rats, and mice has no MHC class I genes or only a few. Most MHC class I genes in non-primate mammalian species are found in other regions. For example, see Ando et al (2005 Immunogenetics 57,864) for the pig alpha, beta, and kappa regions in the MHC class I region. There are no pig MHC genes in the alpha block.

Yes, which is exactly why we use the phrase “in humans” in that particular sentence. The arrangement of the MHC in several other primate reference genomes is shown in Figure 1 - figure supplement 2.

(13) Line 297 to 299. 'The alpha-block also contains a large number of repetitive elements and gene fragments belonging to other gene families, and their specific repeating pattern in humans led to the conclusion that the region was formed by successive block duplications (Shiina et al., 1999).' There are different models for successive block duplications in the alpha block and some are more parsimonious based on imperfect multigenic segmental duplications (Kulski et al 1999, 2000) than others (Shiina et al., 1999). In this regard, Kulski et al (1999, 2000) also used duplicated repetitive elements neighbouring MHC genes to support their phylogenetic analyses and multigenic segmental duplication models. For comparison, can the authors indicate how many duplications and deletions they have in their models for each species?

We have added citations to this sentence to show that there are different published models to describe the successive block duplications (line 307). Our models in Figure 6 and Figure 7 are meant to aggregate past work and integrate our own, and thus they were not built strictly by parsimony. References can be found in Figure 6 - figure supplement 1 and Figure 7 - figure supplement 1.

(14) Lines 315-315. 'Ours is the first work to show that MHC-U is actually an MHC-A-related gene fragment.' This sentence should be deleted. Other researchers had already inferred that MHC-U is actually an MHC-A-related gene fragment more than 25 years ago (Kulski et al 1999, 2000) when the MHC-U was originally named MHC-21.

While these works certainly describe MHC-U/MHC-21 as a fragment in the 𝛼-block, any relation to MHC-A was by association only and very few species/haplotypes were examined. So although the idea is not wholly novel, we provide convincing evidence that not only is MHC-U related to MHC-A by sequence, but also that it is a very recent partial duplicate of MHC-A. We show this with Bayesian phylogenetic trees as well as an analysis of haplotypes across many more species than were included in those papers.  

(15) Lines 361-362. 'Notably, our work has revealed that MHC-V is an old fragment.' This is not a new finding or hypothesis. Previous phylogenetic analysis and gene duplication modelling had already inferred HLA-V (formerly HLA-75) to be an old fragment (Kulski et al 1999, 2000).

By “old,” we mean older than previous hypotheses suggest. Previous work has proposed that MHC-V and -P were duplicated together, with MHC-V deriving from an MHC-A/H/V ancestral gene and MHC-P deriving from an MHC-W/T/P ancestral gene (Kulski (2005), Shiina (1999)). However, our analysis (Figure 5A) shows that MHC-V sequences form a monophyletic clade outside of the MHC-W/P/T group of genes as well as outside of the MHC-A/B/C/E/F/G/J/K/L group of genes, which is not consistent with MHC-A and -V being closely related. Thus, we conclude that MHC-V split off earlier than the differentiation of these other gene groups and is thus older than previously thought. We explain this in the text as well (lines 317-327) and in Appendix 3.  

(16) Line 431-433. 'the Class II genes have been largely stable across the mammals, although we do see some lineage-specific expansions and contractions (Figure 2 and Figure 2-gure Supplement 2).' Please provide one or two references to support this statement. Is 'gure' a typo?

We corrected this typo, thank you! This conclusion is simply drawn from the data presented in Figure 2 and Figure 2 - figure supplement 2. The data itself comes from a variety of sources, which are already included in the supplement as Figure 2 - source data 1.

(17) Line 437. 'We discovered far more "specific" events in Class I, while "broad-scale" events were predominant in Class II.' Please define the difference between 'specific' and 'broad-scale'.

These terms are defined in the previous sentence (lines 466-469).

450-451. 'This shows that classical genes experience more turnover and are more often affected by long-term balancing selection or convergent evolution.' Is balancing selection a form of divergent evolution that is different from convergent evolution? Please explain in more detail how and why balancing selection or convergent evolution affects classical and nonclassical genes differently.

Balancing selection acts to keep alleles at moderate frequencies, preventing any from fixing in the population. In contrast, convergent evolution describes sequences or traits becoming similar over time even though they are not similar by descent. While we cannot know exactly what selective forces have occurred in the past, we observe different patterns in the trees for each type of gene. In Figures 1 and 2, viewers can see at first glance that the nonclassical genes (which are named throughout the text and thoroughly described in Appendix 3) appear to be longer-lived than the classical genes. In addition, lines 204-222 and 475-488 describe topological differences in the BEAST2 trees of these two types of genes. However, we acknowledge that it could be helpful to have additional, complimentary information about the classical vs. non-classical genes. Thus, we have added a sentence and reference to our companion paper (Fortier and Pritchard, 2025), which focuses on long-term balancing selection and draws further contrast between classical and non-classical genes. In lines 481-484, we added  “We further explore the differences between classical and non-classical genes in our companion paper, finding ancient trans-species polymorphism at the classical genes but not at the non-classical genes \citep{Fortier2025b}.”

References

Some references in the supplementary materials such as Alvarez (1997), Daza-Vamenta (2004), Rojo (2005), Aarnink (2014), Kulski (2022), and others are missing from the Reference list. Please check that all the references in the text and the supplementary materials are listed correctly and alphabetically.

We will make sure that these all show up properly in the proof.

Reviewer #3 (Public review):

Summary:

The article provides the most comprehensive overview of primate MHC class I and class II genes to date, combining published data with an exploration of the available genome assemblies in a coherent phylogenetic framework and formulating new hypotheses about the evolution of the primate MHC genomic region.

Strengths:

I think this is a solid piece of work that will be the reference for years to come, at least until population-scale haplotype-resolved whole-genome resequencing of any mammalian species becomes standard. The work is timely because there is an obvious need to move beyond short amplicon-based polymorphism surveys and classical comparative genomic studies. The paper is data-rich and the approach taken by the authors, i.e. an integrative phylogeny of all MHC genes within a given class across species and the inclusion of often ignored pseudogenes, makes a lot of sense. The focus on primates is a good idea because of the wealth of genomic and, in some cases, functional data, and the relatively densely populated phylogenetic tree facilitates the reconstruction of rapid evolutionary events, providing insights into the mechanisms of MHC evolution. Appendices 1-2 may seem unusual at first glance, but I found them helpful in distilling the information that the authors consider essential, thus reducing the need for the reader to wade through a vast amount of literature. Appendix 3 is an extremely valuable companion in navigating the maze of primate MHC genes and associated terminology.

Weaknesses:

I have not identified major weaknesses and my comments are mostly requests for clarification and justification of some methodological choices.

Thank you so much for your kind and supportive review!

Reviewer #1 (Recommendations for the authors):

(1) Line 151: How is 'extensively studied' defined?

Extensively studied is not a strict definition, but a few organisms clearly stand apart from the rest in terms of how thoroughly their MHC regions have been studied. For example, the macaque is a model organism, and individuals from many different species and populations have had their MHC regions fully sequenced. This is in contrast to the gibbon, for example, in which there is some experimental evidence for the presence of certain genes, but no MHC region has been fully sequenced from these animals.

(2) Can you clarify how 'classical' and 'non-classical' MHC genes are being determined in your analysis?

Classical genes are those whose protein products perform antigen presentation to T cells and are directly involved in adaptive immunity, while non-classical genes are those whose protein products do not do this. For example, these non-classical genes might code for proteins that interact with receptors on Natural Killer cells and influence innate immunity. The roles of these proteins are not necessarily conserved between closely related species, and experimental evidence is needed to evaluate this. However, in the absence of such evidence, wherever possible we have provided our best guess as to the roles of the orthologous genes in other species, presented in Figure 1 - source data 1 and Figure 2 - source data 1. This is based on whatever evidence is available at the moment, sometimes experimental but typically based on dN/dS ratios and other indirect measures.

(3) I find the overall tone of the paper to be very descriptive, and at times meandering and repetitive, with a lot of similar kinds of statements being repeated about gene gain/loss. This is perhaps inevitable because a single question is being asked of each of many subsets of MHC gene types, and even exons within gene types, so there is a lot of repetition in content with a slightly different focus each time. This does not help the reader stay focused or keep track. I found myself wishing for a clearly defined question or hypothesis, or some rate parameter in need of estimation. I would encourage the authors to tighten up their phrasing, or consider streamlining the results with some better signposting to organize ideas within the results.

We totally understand your critique, as we talk about a wide range of specific genes and gene groups in this paper. To improve readability, we have added many more signposting phrases and sentences:

“Aside from MHC-DRB, …” (line 173)

“Now that we had a better picture of the landscape of MHC genes present in different primates, we wanted to understand the genes’ relationships. Treating Class I, Class IIA, and Class IIB separately, ...” (line 179-180)

“We focus first on the Class I genes.” (line 191)

“... for visualization purposes…” (line195)

“We find that sequences do not always assort by locus, as would be expected for a typical gene.” (lines 196-197)

“... rather than being directly orthologous to the ape/OWM MHC-G genes.” (lines 201-202)

“Appendix 3 explains each of these genes in detail, including previous work and findings from this study.“ (lines 202-203)

“... (but not with NWM) …” (line 208)

“While genes such as MHC-F have trees which closely match the overall species tree, other genes show markedly different patterns, …” (lines 212-213)

“Thus, while some MHC-G duplications appear to have occurred prior to speciation events within the NWM, others are species-specific.” (lines 218-219)

“... indicating rapid evolution of many of the Class I genes” (lines 220-221)

“Now turning to the Class II genes, …“ (line 223)

“(see Appendix 2 for details on allele nomenclature) “ (line 238)

“(e.g. MHC-DRB1 or -DRB2)” (line 254)

“...  meaning their names reflect previously-observed functional similarity more than evolutionary relatedness.” (lines 257-258)

“(see Appendix 3 for more detail)” (line 311)

“(a 5'-end fragment)” (line 324)

“Therefore, we support past work that has deemed MHC-V an old fragment.” (lines 326-327)

“We next focus on MHC-U, a previously-uncharacterized fragment pseudogene containing only exon 3.” (line 328-329)

“However, it is present on both chimpanzee haplotypes and nearly all human haplotypes, and we know that these haplotypes diverged earlier---in the ancestor of human and gorilla. Therefore, ...” (lines 331-333)

“Ours is the first work to show that MHC-U is actually an MHC-A-related gene fragment and that it likely originated in the human-gorilla ancestor.” (lines 334-336)  

“These pieces of evidence suggest that MHC-K and -KL duplicated in the ancestor of the apes.” (lines 341-342)

“Another large group of related pseudogenes in the Class I $\alpha$-block includes MHC-W, -P, and -T (see Appendix 3 for more detail).” (lines 349-350)

“...to form the current physical arrangement” (lines 354)

“Thus, we next focus on the behavior of this subgroup in the trees.” (line 358)

“(see Appendix 3 for further explanation).” (line 369)

“Thus, for the first time we show that there must have been three distinct MHC-W-like genes in the ape/OWM ancestor.” (lines 369-371)

“... and thus not included in the previous analysis. ” (lines 376-377)

“MHC-Y has also been identified in gorillas (Gogo-Y) (Hans et al., 2017), so we anticipate that Gogo-OLI will soon be confirmed. This evidence suggests that the MHC-Y and -OLI-containing haplotype is at least as old as the human-gorilla split. Our study is the first to place MHC-OLI in the overall story of MHC haplotype evolution“ (lines 381-384)

“Appendix 3 explains the pieces of evidence leading to all of these conclusions (and more!) in more detail.” (lines 395-396)

“However, looking at this exon alone does not give us a complete picture.” (lines 410-411)

“...instead of with other ape/OWM sequences, …” (lines 413-414)

“Figure 7 shows plausible steps that might have generated the current haplotypes and patterns of variation that we see in present-day primates. However, some species are poorly represented in the data, so the relationships between their genes and haplotypes are somewhat unclear.” (lines 427-429)

“(and more-diverged)” (line 473)

“(of both classes)” (line 476)

“..., although the classes differ in their rate of evolution.”  (line 487-488)

“Including these pseudogenes in our trees helped us construct a new model of $\alpha$-block haplotype evolution. “ (lines 517-518)

(4) Line 480-82: "Notably...." why is this notable? Don't merely state that something is notable, explain what makes it especially worth drawing the reader's attention to: in what way is it particularly significant or surprising?

We have changed the text from “Notably” to “In particular” (line 390) so that readers are expecting us to list some specific findings. Similarly, we changed “Notably” to “Specifically” (line 515).

(5) The end of the discussion is weak: "provide context" is too vague and not a strong statement of something that we learned that we didn't know before, or its importance. This is followed by "This work will provide a jumping-off point for further exploration..." such as? What questions does this paper raise that merit further work?

We have made this paragraph more specific and added some possible future research directions. It now reads “By treating the MHC genes as a gene family and including more data than ever before, this work enhances our understanding of the evolutionary history of this remarkable region. Our extensive set of trees incorporating classical genes, non-classical genes, pseudogenes, gene fragments, and alleles of medical interest across a wide range of species will provide context for future evolutionary, genomic, disease, and immunologic studies. For example, this work provides a jumping-off-point for further exploration of the evolutionary processes affecting different subsets of the gene family and the nuances of immune system function in different species. This study also provides a necessary framework for understanding the evolution of particular allelic lineages within specific MHC genes, which we explore further in our companion paper \citep{Fortier2025b}. Both studies shed light on MHC gene family evolutionary dynamics and bring us closer to understanding the evolutionary tradeoffs involved in MHC disease associations.” (lines 576-586)

Reviewer #3 (Recommendations for the authors):

(1) Figure 1 et seq. Classifying genes as having 'classical', 'non-classical' and 'dual' properties is notoriously difficult in non-model organisms due to the lack of relevant information. As you have characterised a number of genes for the first time in this paper and could not rely entirely on published classifications, please indicate the criteria you used for classification.

The roles of these proteins are not necessarily conserved between closely related species, and experimental evidence is needed to evaluate this. However, in the absence of such evidence, wherever possible we have provided our best guess as to the roles of the orthologous genes in other species, presented in Figure 1 - source data 1 and Figure 2 - source data 1. This is based on whatever evidence is available at the moment, sometimes experimental but typically based on dN/dS ratios and other indirect measures.

(2) Line 61 It's important to mention that classical MHC molecules present antigenic peptides to T cells with variable alphabeta T cell receptors, as non-classical MHC molecules may interact with other T cell subsets/types.

Thank you for pointing this out; we have updated the text to make this clearer (lines 63-65). We changed “‘Classical’ MHC molecules perform antigen presentation to T cells---a key part of adaptive immunity---while ‘non-classical’ molecules have niche immune roles.” to “‘Classical’ MHC molecules perform antigen presentation to T cells with variable alphabeta TCRs---a key part of adaptive immunity---while ‘non-classical’ molecules have niche immune roles.”

(3) Perhaps it's worth mentioning in the introduction that you are deliberately excluding highly divergent non-classical MHC molecules such as CD1.

Thank you, it’s worth clarifying exactly what molecules we are discussing. We have added a sentence to the introduction (lines 38-43): “Having originated in the jawed vertebrates, this group of genes is now involved in diverse functions including lipid metabolism, iron uptake regulation, and immune system function (proteins such as zinc-𝛼2-glycoprotein (ZAG), human hemochromatosis protein (HFE), MHC class I chain–related proteins (MICA, MICB), and the CD1 family) \citep{Hansen2007,Kupfermann1999,Kaufman2022,Adams2013}. However, here we focus on…”

(4) Line 94-105 This material presents results, it could be moved to the results section as it now somewhat disrupts the flow.

We feel it is important to include a “teaser” of the results in the introduction, which can be slightly more detailed than that in the abstract.

(5) Line 118-131 This opening section of the results sets the stage for the whole presentation and contains important information that I feel needs to be expanded to include an overview and justification of your methodological choices. As the M&M section is at the end of the MS (and contains limited justification), some information on two aspects is needed here for the benefit of the reader. First, as far as I understand, all phylogenetic inferences were based entirely on DNA sequences of individual (in some cases concatenated) exons. It would be useful for the reader to explain why you've chosen to rely on DNA rather than protein sequences, even though some of the genes you include in the phylogenetic analysis are highly divergent. Second, a reader might wonder how the "maximum clade credibility tree" from the Bayesian analysis compares to commonly seen trees with bootstrap support or posterior probability values assigned to particular clades. Personally, I think that the authors' approach to identifying and presenting representative trees is reasonable (although one might wonder why "Maximum clade credibility tree" and not "Maximum credibility tree" https://www.beast2.org/summarizing-posterior-trees/), since they are working with a large number of short, sometimes divergent and sometimes rather similar sequences - in such cases, a requirement for strict clade support could result in trees composed largely of polytomies. However, I feel it's necessary to be explicit about this and to acknowledge that the relationships represented by fully resolved bifurcating representative trees and interpreted in the study may not actually be highly supported in the sense that many readers might expect. In other words, the reader should be aware from the outset of what the phylogenies that are so central to the paper represent.

We chose to rely on DNA rather than protein sequences because convergent evolution is likely to happen in regions that code for extremely important functions such as adaptive and innate immunity. Convergent evolution acts upon proteins while trans-species polymorphism retains ancient nucleotide variation, so studying the DNA sequence can help tease apart convergent evolution from trans-species polymorphism.

As for the “maximum clade credibility tree”, this is a matter of confusing nomenclature. In the online reference guide (https://www.beast2.org/summarizing-posterior-trees/), the tree with the maximum product of the posterior clade probabilities is called the “maximum credibility tree” while the tree that has the maximum sum of posterior clade probabilities is called the “Maximum credibility tree”. The “Maximum credibility tree” (referring to the sum) appears to have only been named in this way in the first version of TreeAnnotator. However, the version of TreeAnnotator that I used lists the options “maximum clade credibility tree” and “maximum sum of clade probabilities”. So the context suggests that the “maximum clade credibility tree” option is actually maximizing the product. This “maximum clade credibility tree” is the setting I used for this project (in TreeAnnotator version 2.6.3).

We agree that readers may not fully grasp what the collapsed trees represent upon first read. We have added a sentence to the beginning of the results (line 188-190) to make this more explicit.

(6) Line 224, you're referring to the DPB1*09 lineage, not the DRB1*09 lineage.

Indeed! We have changed these typos.

(7) Line 409, why "Differences between MHC subfamilies" and not "Differences between MHC classes"?

We chose the word “subfamilies” because we discuss the difference between classical and non-classical genes in addition to differences between Class I and Class II genes.

(8) Line 529-544 This might work better as a table.

We agree! This information is now presented as Table 1.

(9) Line 547 MHC-DRB9 appears out of the blue here - please say why you are singling it out.

Great point! We added a paragraph (lines 614-623) to explain why this was necessary.

(10) Line 550-551 Even though you've screened the hits manually, it would be helpful to outline your criteria for this search.

Thank you! We’ve added a couple of sentences to explain how we did this (lines 607-610).

(11) Line 556-580 please provide nucleotide alignments as supplementary data so that the reader can get an idea of the actual divergence of the sequences that have been aligned together.

Thank you! We’ve added nucleotide alignments as supplementary files.

(12) Line 651-652 Why "Maximum clade credibility tree" and not "Maximum credibility tree"? 

Repeat of (5). This is a matter of confusing nomenclature. In the online reference guide (https://www.beast2.org/summarizing-posterior-trees/), the tree with the maximum product of the posterior clade probabilities is called the “maximum credibility tree” while the tree that has the maximum sum of posterior clade probabilities is called the “Maximum credibility tree”. The “Maximum credibility tree” (referring to the sum) appears to have only been named in this way in the first version of TreeAnnotator. However, the version of TreeAnnotator that I used lists the options “maximum clade credibility tree” and “maximum sum of clade probabilities”. So the context suggests that the “maximum clade credibility tree” option is actually maximizing the product. This “maximum clade credibility tree” is the setting I used for this project (in TreeAnnotator version 2.6.3).

(13) In the appendices, links to references do not work as expected.

We will make sure these work properly when we receive the proofs.

Note de synthèse : Les formes de la violence et le témoignage

Ce document de synthèse explore les différentes formes et fonctions du témoignage face à la violence, en s'appuyant sur l'analyse de Didier Fassin dans "Les formes de la violence (8)".

Il met en lumière l'importance de l'attestation de la violence, les diverses figures du témoin, les défis de sa représentation, et l'émergence de nouvelles médiations technologiques pour révéler la vérité.

I. L'attestation de la violence : une urgence face à l'invisibilisation

La raison d'être la plus commune de l'écriture et de la représentation de la violence est de l'attester, une urgence d'autant plus grande que la réalité est invisibilisée. L'auteur cite deux exemples contemporains de cette invisibilisation et des tentatives d'attestation :

La violence coloniale française en Algérie : Malgré une loi de 2005 qui "oblige les programmes scolaires... à reconnaître le rôle positif de la présence française outre-mer", des travaux comme celui d'Alain Ruot (2024) dans "La première guerre en Algérie" rappellent les "spoliations de terre, les déplacements de population, les massacres de villageois, les enfumades de grottes, les centaines de milliers de morts surtout des civils" perpétrées par le corps expéditionnaire français.

L'expulsion des Palestiniens (la Nakba) : L'expulsion de "750 000 Palestiniens, soit environ la moitié de la population arabe de ce territoire", qui a entraîné la "destruction de villages et dans certains cas du meurtre de leurs habitants", a longtemps été ignorée.

Le film "Partition" (2025) de Dana Alan, prolongeant son ouvrage "Voices of the Nagba", vise à "restituer l'expérience de l'enagbactrale à travers les archives coloniales du mandat britannique" et les récits des Palestiniens.

Ces entreprises visent à attester ce que les nations ont "enfoui souvent dans les profondeurs de l'oubli".

Si les auteurs de violence peuvent avoir intérêt à la montrer pour "la jouissance de l'exercice de la force à la production d'un régime de terreur", ils ont souvent "un intérêt plus grand encore à la dissimuler, à la déguiser, à la nier" pour éviter la condamnation ou la sanction.

Dans ces cas, il est crucial pour les victimes, leurs proches, et les "entrepreneurs de justice" (avocats, militants des droits humains, chercheurs) d'apporter la preuve de la violence, ses circonstances et ses responsables.

"Attester la violence c'est donc combattre le déni, l'occultation, le mensonge, le révisionnisme historique. Attester la violence c'est emporter témoignage, c'est sans faire le témoin."

II. Les figures du témoin : entre objectivité et subjectivité S'appuyant sur Émile Benveniste, l'auteur distingue deux conceptions du témoin, principalement à travers le latin :

Testis : "celui qui assiste entière à une affaire où deux personnages sont intéressés ayant été présent au moment où les faits se sont produits".

Sa parole "peut être utilisé pour trancher un litige à condition qu'il soit établi qu'il n'était pas lui-même partie prenante". Le testis est extérieur à la scène, son observation est présumée objective.

Superstess : "décrit le témoin comme celui qui subsiste au-delà, témoin en même temps que survivant".

Son témoignage est autorisé par le fait d'avoir "vécu lui-même les faits notamment lorsqu'il s'implique un danger ou une épreuve et d'avoir survécu à ce péril".

Le superstess est la victime, son récit est nécessairement subjectif, mais non insoupçon.

Cette distinction est mise à l'épreuve par la littérature sur la Shoah.

A. Le défi du témoignage face à la dissimulation nazie

L'histoire de l'extermination des Juifs et des Roms n'est pas quelque chose dont les nazis se vantaient, mais qu'ils ont cherché à dissimuler, y compris "vis-à-vis du peuple allemand et vis-à-vis d'eux-mêmes".

Hannah Arendt, dans "Eichmann à Jérusalem", souligne l'usage d'un "langage codé" ou "règles de langage" qui étaient "dans le parler ordinaire... un mensonge", pour euphémiser les crimes : "solution finale", "traitement spécial", "évacuation".

L'effet de ce système de langage n'était pas "d'empêcher les gens de savoir ce qu'ils faisaient, mais de les empêcher de mettre leurs actes en rapport avec leur ancienne notion normale du meurtre et du mensonge, en somme de rendre mentalement acceptable ce qui aurait pu leur paraître moralement intolérable."

Pierre Vidal-Naquet ajoute que ce langage codé a facilité le négationnisme ultérieur.

Les nazis, conscients de ce qui allait se passer, avertissaient cyniquement les prisonniers : "De quelque façon que cette guerre se finisse, nous l'avons déjà gagné contre vous ; aucun d'entre vous ne restera pour porter témoignage.

Mais même si quelques-uns en réchappaient, le monde ne les croira pas, il n'y aura pas de certitude, car nous détruirons les preuves en vous détruisant." (Primo Levi, "Les naufragés et les rescapés").

Cette peur du non-crédit a hanté les survivants, qui ont souvent raconté un cauchemar récurrent où leurs proches ne les croyaient pas.

D'où l'importance vitale du témoignage, comme l'exprime Robert Antelme : "nous voulions parler, être entendu enfin".

B. La complexité du témoignage des survivants (Superstess/Testis)

Primo Levi, en écrivant "Si c'est un homme", cherchait à "attester" son expérience.

Cependant, il exprime une profonde gêne, estimant que "nous les survivants ne sommes pas les vrais témoins... car nous sommes ceux qui grâce à la prévarication, l'habileté ou la chance, n'ont pas touché le fond."

Les "musulmans" (ceux tellement affaiblis qu'ils étaient voués à mourir) sont les "témoins intégraux".

La réflexion de Levi met à l'épreuve la distinction testis/superstess :

• Il est un superstess incontestable, ayant survécu à l'impensable et décrivant l'insulte de la "démolition d'un homme".
• Mais il est aussi un testis, conscient de ne jamais pouvoir restituer l'expérience de ceux qui ont été dévorés, et pour qui il parle "à leur place, par délégation".

L'exemple d'Urbinec, l'enfant paralysé et mutique à Auschwitz, dont la "nécessité de parler jaillissait dans son regard avec une force explosive", et dont Primo Levi écrit "il témoigne à travers mes paroles", illustre cette réconciliation tragique des deux figures : "le superstès devenu testis sauve du néant la mémoire du petit garçon."

C. Diversité des styles et temporalités du témoignage

Les récits des survivants du génocide adoptent des styles et des temporalités variés :

• Témoignage immédiat : David Rousset ("L'univers concentrationnaire", 1946) rencontre un succès rapide malgré la réticence des sociétés européennes, peut-être grâce à une "forme de recherche esthétique" créant une distance "qui neutralise les émotions".

Son écriture est "austère et ironique", utilisant "des formules elliptiques et tranchantes, parfois caustiques et troublantes."

• Témoignage différé : Charlotte Delbo ("Aucun de nous ne reviendra", 1965), écrit un premier brouillon après sa sortie, puis le reprend 20 ans plus tard. Elle commence par la scène collective des arrivées de trains, utilisant des phrases courtes et des images fortes pour dire "l'inconcevable".

• Anti-mémoire : Imre Kertész ("Être et destin", 1985) adopte le regard "naïf déconcerté" d'un adolescent, décrivant la découverte progressive de l'horreur des camps, comme "l'odeur... doucâtre, en quelque sorte gluante" du crématorium.

Il décrit la "détérioration physique" sans pathos, et même un "désir sourd" de vivre au moment du "tri final des mourants".

• Méfiance et refus d'enfermement : Ruth Kluger ("Refus de témoigner. Une jeunesse", 1992) écrit pour exprimer sa méfiance face à la multiplication des témoignages et son refus d'être réduite à sa condition de déportée.

• L'expérience des victimes du nazisme est à la fois "spécifique" (partir d'un vécu individuel) et "indéterminée" (nécessité de trouver les mots et la forme face à "l'incommunicabilité abyssale").

Pour l'immense majorité des survivants, il faut "accepter de n'être ni superstès ni testice et donc se taire."

III. Autres figures du témoin et médiations

A. Auctor et Histor : l'autorité et la connaissance

Auctor (latin) : "celui qui augmente la confiance, le garant, la source et donc l'autorité" et "celui qui pousse à agir, l'instigateur, le créateur et donc l'auteur".

Le crédit est le fondement de son témoignage.

Histor (grec) : "celui qui sait, qui connaît... l'historien". L'enquête est le fondement de son témoignage.

Ces figures n'ont pas vécu les faits mais peuvent en être les garants. Les historiens contemporains "réunissent souvent les deux dimensions", bénéficiant du "crédit de leur discipline" et s'appuyant sur des "enquêtes menées dans des archives ou par des entretiens".

L'exemple de Jean Hatzfeld et son livre "Dans le nu de la vie" (2000) sur le génocide rwandais illustre l'auctor.

Il rassemble des récits de survivants, s'autorisant à les convaincre de parler malgré leur réticence.

Journaliste et écrivain, il utilise sa double autorité pour "attester ce qu'a été et ce qu'est encore... l'expérience de ces hommes, de ces femmes, de ces enfants qui ont vécu le massacre."

Bien que les récits soient rédigés à la première personne, ils sont "entièrement écrits par une troisième personne, l'auteur."

• L'histore est illustré par les chercheurs en sciences sociales qui restituent et interprètent les faits en s'appuyant sur des "archives nationales ou étrangères, des jugements rendus par des juridictions internationales, des articles de journaux locaux, des entretiens avec des personnes occupant des positions différentes, des observations de procès".

Les travaux de Mahmoud Mamdani ("When Victims Become Killers", 2001) interprètent le génocide rwandais à la lumière de l'histoire coloniale, distinguant le génocide conduit par les "settlers" (colons) et celui par les "natives" (indigènes).

Hélène Dumas ("Le génocide au village", 2014) se concentre sur la "mécanique microlocale des violences", montrant que le génocide est "une affaire de voisins et de parents" et que les génocidaires "éprouvent une jouissance dans la souffrance et l'humiliation de leurs victimes."

Beata Umubyeyi Mairesse ("Le convoi", 2024), une survivante du génocide rwandais, se distingue par sa réflexivité et son intégrité.

Elle est à la fois superstess, racontant sa survie, et testis, décrivant ce qu'elle a vu.

Elle se fait également historienne de son histoire, explorant des archives et conduisant des entretiens, mais "elle répugne à faire acte d'autorité," refusant d'être l'auctor.

B. Martous : le témoin-martyr

En grec ancien, "Martous" signifie le témoin, mais aussi, plus spécifiquement dans la Bible, le "témoin de Dieu", c'est-à-dire le martyr, celui qui "a accepté de mourir pour attester de sa croyance".

Giorgio Agamben ("Ce qui reste d'Auschwitz", 1998) note que le martyre chrétien a dû "justifier le scandale d'une mort insensée".

Le "shaï" arabe a un sens similaire, désignant à la fois le témoin et le martyr.

En Palestine, la figure du shaïd s'est développée comme "ciment de l'unité nationale".

Le shaïd peut être une victime tuée "sans l'avoir choisi" ou un combattant qui s'est exposé "volontairement pour la cause de son peuple".

Ce dédoublement transforme le sens du martyre, l'étendant du "sacrifice librement consenti à la mort subie", et du "strictement religieux au politique".

"Tout palestinien abattu ou exécuté par les Israéliens est un shaïd qui par sa mort dans un affrontement inégal atteste son appartenance à sa communauté et témoigne de la brutalisation de l'ennemi."

Pour les martyrs palestiniens, le sacrifice ou la mort est une réponse à une "vie impossible à quoi la mort viendrait tragiquement redonner du sens".

L'auteur cite la photojournaliste Fatima Assuna : "Quant à la mort qui est inévitable, si je meurs, je veux une mort retentissante, je ne veux pas être une simple brève dans un flash info ni un chiffre parmi d'autres, je veux une mort dont le monde entier entendra parler, une empreinte qui restera à jamais, des émotions, des images immortelles que ni le temps ni l'espace ne pourront enterrer."

IV. Les médiations technologiques du témoignage

Le témoignage ne s'exprime pas seulement par la parole, l'écrit ou le corps (dans le cas du martyr), mais aussi par des "médiations dans lesquelles les technologies peuvent être mobilisées".

L'exemple le plus innovant est Forensic Architecture (fondée en 2010 par Eyal Weizman), une agence qui développe des "techniques, méthodes et concepts pour conduire des investigations sur la violence d'État et la violence en entreprise".

• En combinant "l'imagerie spatiale par satellite, les caméras de surveillance, les enregistrements audio et vidéo, les témoignages individuels et collectifs", Forensic Architecture reconstitue en 3D des événements de violence qui ont été occultés.

Parmi les nombreux cas étudiés, on trouve le génocide des Herero et Nama, les massacres israéliens pendant la Nakba, l'assassinat d'otages en Colombie, le meurtre de Mark Duggan au Royaume-Uni, l'utilisation d'armes européennes au Yémen, et des événements en France (Adama Traoré, Zineb Reddouane).

Ces technologies permettent de "révéler de nombreuses violences, des crimes de guerre identifiés, des coupables reconnus, des versions officielles démenties, certaines vérités dites et la justice parfois rendue".

Elles "renforcent, enrichissent et parfois même remplacent le témoignage humain".

V. Conclusion : La complexité du témoignage pour faire exister la vérité

En résumé, l'auteur a esquissé cinq figures idéaltypiques du témoin :

• Le testis : présent au moment des faits, dont il peut raconter.
• Le superstess : survivant, qui peut transmettre ce qu'il a vécu.
• L'auctor : agent extérieur, qui apporte la crédibilité.
• L'histor : expert légitime, qui conduit une enquête.

• Le martous : victime sacrificielle, qui affirme la justesse de sa cause par son renoncement.

• Chacune de ces figures "engage des formes politiques et morales : la véracité du testis, l'authenticité du superstès, l'autorité de l'actor, la neutralité de l'histor, l'engagement du Martus."

Ces figures ne sont pas étanches et "se mêlent, se combinent, se déplacent, se complexifient" dans la réalité.

Au-delà de ces distinctions, "l'enjeu du témoignage c'est de faire exister une vérité et notamment... de la faire exister contre la dissimulation, l'invisibilisation, la dénégation".

C'est là toute l'importance de "celles et ceux qui ont pour projet de révéler la vérité ou tout au moins une part de la vérité à laquelle ils ont eu accès."

eLife Assessment

This study provides important insights into how researchers can use perceptual metamers to formally explore the limits of visual representations at different processing stages. The framework is compelling and the data largely support the claims, subject to minor caveats.

Reviewer #1 (Public review):

This is an interesting study on the nature of representations across the visual field. The question of how peripheral vision differs from foveal vision is a fascinating and important one. The majority of our visual field is extra-foveal yet our sensory and perceptual capabilities decline in pronounced and well-documented ways away from the fovea. Part of the decline is thought to be due to spatial averaging ('pooling') of features. Here, the authors contrast two models of such feature pooling with human judgments of image content. They use much larger visual stimuli than in most previous studies, and some sophisticated image synthesis methods to tease apart the prediction of the distinct models.

More importantly, in so doing, the researchers thoroughly explore the general approach of probing visual representations through metamers-stimuli that are physically distinct but perceptually indistinguishable. The work is embedded within a rigorous and general mathematical framework for expressing equivalence classes of images and how visual representations influence these. They describe how image-computable models can be used to make predictions about metamers, which can then be compared to make inferences about the underlying sensory representations. The main merit of the work lies in providing a formal framework for reasoning about metamers and their implications, for comparing models of sensory processing in terms of the metamers that they predict, and for mapping such models onto physiology. Importantly, they also consider the limits of what can be inferred about sensory processing from metamers derived from different models.

Overall, the work is of a very high standard and represents a significant advance over our current understanding of perceptual representations of image structure at different locations across the visual field. The authors do a good job of capturing the limits of their approach I particularly appreciated the detailed and thoughtful Discussion section and the suggestion to extend the metamer-based approach described in the MS with observer models. The work will have an impact on researchers studying many different aspects of visual function including texture perception, crowding, natural image statistics and the physiology of low- and mid-level vision.

The main weaknesses of the original submission relate to the writing. A clearer motivation could have been provided for the specific models that they consider, and the text could have been written in a more didactic and easy to follow manner. The authors could also have been more explicit about the assumptions that they make.

Comments following re-submission:

Overall, I think the authors have done a satisfactory job of addressing most of the points I raised.

There's one final issue which I think still needs better discussion.

I think reviewer 2 articulated better than I have the point I was concerned about: the relationship between JNDs and metamers as depicted in the schematics and indeed in the whole conceptualization.

I think the issue here is that there seems to be a conflating of two concepts- 'subthreshold' and 'metamer'-and I'm not convinced it is entirely unproblematic. It's true that two stimuli that cannot be discriminated from one another due to the physical differences being too small to detect reliably by the visual system are a form of metamer in the strict definition 'physically different, but perceptually the same'.<br /> However, I don't think this is the scientifically substantial notion of metamer that enabled insights into trichromacy. That form of metamerism is due to the principle of univariance in feature encoding, and involves conditions in which physically very different stimuli are mapped to one and the same point in sensory encoding space whether or not there is any noise in the system. When I say 'physically very different' I mean different by a large enough amount that they would be far above threshold, potentially orders of magnitude larger than a JND if the system's noise properties were identical but the system used a different sensory basis set to measure them. This seems to be a very different kind of 'physically different, but perceptually the same'.

I do think the notion of metamerism can obviously be very usefully extended beyond photoreceptors and photon absorptions. In the interesting case of texture metamers, what I think is meant is that stimuli would be discriminable if scrutinised in the fovea, but because they have the same statistics they are treated as equivalent. I think the discussion of this could still be clearly articulated in the manuscript. It would benefit from a more thorough discussion of the difference between metamerism and subthreshold, especially in the context of the Voronoi diagrams at the beginning.

It needs to be made clear to the reader why it is that two stimuli that are physically similar (e.g., just spanning one of the edges in the diagram) can be discriminable, while at the same time, two stimuli that are very different (e.g., at opposite ends of a cell) can't.

Do the cells include BOTH those sets of stimuli that cannot be discriminated just because of internal noise AND those that can't be discriminated because they are projected to literally the same point in the sensory encoding space? What are the strengths and limits of models that involve the strict binarization of sensory representations, and how can they be integrated with models dealing with continuous differences? These seem like important background concepts that ought to be included in either the introduction of discussion sections. In this context it might also be helpful to refer to the notion of 'visual equivalence' as described by:

Ramanarayanan, G., Ferwerda, J., Walter, B., & Bala, K. (2007). Visual equivalence: towards a new standard for image fidelity. ACM Transactions on Graphics (TOG), 26(3), 76-es.

Other than that, I congratulate the authors on a very interesting study, and look forward to reading the final version.

Reviewer #2 (Public review):

Summary:

The authors have improved clarity overall and have spoken to most of the issues raised by the reviewers. There are still two outstanding problems however, where issues raised during the review were inappropriately dismissed in the manuscript. These should be explicitly addressed as limitations to the results presented (no eye tracking), and early pilot experiments that informed the experiments as presented (pink noise) rather than brushed off as 'unnecessary' and 'would be uninformative'.

Eye tracking:

It is generally accepted that experiments testing stimuli presented at specific locations in peripheral vision require eye tracking to ensure that the stimulus is presented as expected, in particular, in the correct location. As I stated in the previous round of review, while a stimulus presentation time of 200ms does help eliminate some saccades, it does not eliminate the possibility that subjects were not fixating well during stimulus onset. I am also unclear what the authors mean by 'trained observer' in this context, though the authors state that an author subject in a different portion of the paper is an 'expert observer'. Does this mean the 'trained observers' are non-expert recruited subjects? Given the conditions tested differ from previous work (Freeman & Simoncelli, 2011) *these differences are a main contribution of the paper!* which DID include eye tracking in a subset of subjects, it is entirely possible to get similar results to this work in the context of non eye-tracking controlled stimulus presentation. The reasons now in the manuscript are not reasons that make eye tracking 'considered unnecessary'.

I appreciate that the authors now state the lack of eye tracking explicitly, but believe the paper needs to at least state that this is a limitation of the results reported, and eyetracking being 'considered unnecessary' is unreasonable, nor a norm in this subfield.

N=1: The authors now state clearly the limitations of a single subject in the manuscript, and state the expertise level of this subject.

Large number of trials: The authors now address this and include an enumeration of the large number of trials.

Simple Models / Physiology comparison: I support the choice to reduce claims regarding tight connections to physiology, and appreciate the explanation of the luminance model.

Previous Work: I appreciate the author's changes to the introduction, both in discussing previous work and citation fixes.

Blurred White, Pink Noise: While the authors now address pink noise, the explanation for such stimuli being expected to be uninformative is confusing to me. The manuscript now first states that pink noise is a natural choice, then claims it would be uninformative, while also stating in the rebuttal (not the manuscript) that they tried it and it indeed reduced the artifacts they note. The logic of the experiments indeed relies on finding the smallest critical scaling value, which is measured by subjects determining if a synthesis is similar or different to a target or second synth. A synthesis free from artifacts would surely affect the subjects responses and the smallest critical scaling measured.

The statement that the authors experimented with pink noise early on and found this able to address the artifacts should be stated in the manuscript itself, not just in the rebuttal, and the blanket statement that this experiment would be 'uninformative' is incorrect. Surely this early pilot the authors mention in the rebuttal was informative to designing the experiments that appear in the final paper, and would be an informative experiment to include.

Author response:

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

Reviewer #1 (Public Review):

This is an interesting study of the nature of representations across the visual field. The question of how peripheral vision differs from foveal vision is a fascinating and important one. The majority of our visual field is extra-foveal yet our sensory and perceptual capabilities decline in pronounced and well-documented ways away from the fovea. Part of the decline is thought to be due to spatial averaging (’pooling’) of features. Here, the authors contrast two models of such feature pooling with human judgments of image content. They use much larger visual stimuli than in most previous studies, and some sophisticated image synthesis methods to tease apart the prediction of the distinct models.

More importantly, in so doing, the researchers thoroughly explore the general approach of probing visual representations through metamers-stimuli that are physically distinct but perceptually indistinguishable. The work is embedded within a rigorous and general mathematical framework for expressing equivalence classes of images and how visual representations influence these. They describe how image-computable models can be used to make predictions about metamers, which can then be compared to make inferences about the underlying sensory representations. The main merit of the work lies in providing a formal framework for reasoning about metamers and their implications, for comparing models of sensory processing in terms of the metamers that they predict, and for mapping such models onto physiology. Importantly, they also consider the limits of what can be inferred about sensory processing from metamers derived from different models.

Overall, the work is of a very high standard and represents a significant advance over our current understanding of perceptual representations of image structure at different locations across the visual field. The authors do a good job of capturing the limits of their approach and I particularly appreciated the detailed and thoughtful Discussion section and the suggestion to extend the metamer-based approach described in the MS with observer models. The work will have an impact on researchers studying many different aspects of visual function including texture perception, crowding, natural image statistics, and the physiology of low- and mid-level vision.

The main weaknesses of the original submission relate to the writing. A clearer motivation could have been provided for the specific models that they consider, and the text could have been written in a more didactic and easy-to-follow manner. The authors could also have been more explicit about the assumptions that they make.

Thank you for the summary. We appreciate the positives noted above. We address the weaknesses point by point below.

Reviewer #2 (Public Review):

Summary

This paper expands on the literature on spatial metamers, evaluating different aspects of spatial metamers including the effect of different models and initialization conditions, as well as the relationship between metamers of the human visual system and metamers for a model. The authors conduct psychophysics experiments testing variations of metamer synthesis parameters including type of target image, scaling factor, and initialization parameters, and also compare two different metamer models (luminance vs energy). An additional contribution is doing this for a field of view larger than has been explored previously

General Comments

Overall, this paper addresses some important outstanding questions regarding comparing original to synthesized images in metamer experiments and begins to explore the effect of noise vs image seed on the resulting syntheses. While the paper tests some model classes that could be better motivated, and the results are not particularly groundbreaking, the contributions are convincing and undoubtedly important to the field. The paper includes an interesting Voronoi-like schematic of how to think about perceptual metamers, which I found helpful, but for which I do have some questions and suggestions. I also have some major concerns regarding incomplete psychophysical methodology including lack of eye-tracking, results inferred from a single subject, and a huge number of trials. I have only minor typographical criticisms and suggestions to improve clarity. The authors also use very good data reproducibility practices.

Thank you for the summary. We appreciate the positives noted above. We address the weaknesses point by point below.

Specific Comments

Experimental Setup

Firstly, the experiments do not appear to utilize an eye tracker to monitor fixation. Without eye tracking or another manipulation to ensure fixation, we cannot ensure the subjects were fixating the center of the image, and viewing the metamer as intended. While the short stimulus time (200ms) can help minimize eye movements, this does not guarantee that subjects began the trial with correct fixation, especially in such a long experiment. While Covid-19 did at one point limit in-person eye-tracked experiments, the paper reports no such restrictions that would have made the addition of eye-tracking impossible. While such a large-scale experiment may be difficult to repeat with the addition of eye tracking, the paper would be greatly improved with, at a minimum, an explanation as to why eye tracking was not included.

Addressed on pg. 25, starting on line 658.

Secondly, many of the comparisons later in the paper (Figures 9,10) are made from a single subject. N=1 is not typically accepted as sufficient to draw conclusions in such a psychophysics experiment. Again, if there were restrictions limiting this it should be discussed. Also (P11) Is subject sub-00 is this an author? Other expert? A naive subject? The subject’s expertise in viewing metamers will likely affect their performance.

Addressed on pg. 14, starting on line 308.

Finally, the number of trials per subject is quite large. 13,000 over 9 sessions is much larger than most human experiments in this area. The reason for this should be justified.

In general, we needed a large number of trials to fit full psychometric functions for stimuli derived for both models, with both types of comparison, both initializations, and over many target images. We could have eliminated some of these, but feel that having a consistent dataset across all these conditions is a strength of the paper.

In addition to the sentence on pg. 14, line 318, a full enumeration of trials is now described on pg. 23, starting on line 580.

Model

For the main experiment, the authors compare the results of two models: a ’luminance model’ that spatially pools mean luminance values, and an ’energy model’ that spatially pools energy calculated from a multi-scale pyramid decomposition. They show that these models create metamers that result in different thresholds for human performance, and therefore different critical scaling parameters, with the basic luminance pooling model producing a scaling factor 1/4 that of the energy model. While this is certain to be true, due to the luminance model being so much simpler, the motivation for the simple luminance-based model as a comparison is unclear.

The use of simple models is now addressed on pg. 3, starting on line 98, as well as the sentence starting on pg. 4 line 148: the luminance model is intended as the simplest possible pooling model.

The authors claim that this luminance model captures the response of retinal ganglion cells, often modeled as a center-surround operation (Rodieck, 1964). I am unclear in what aspect(s) the authors claim these center-surround neurons mimic a simple mean luminance, especially in the context of evidence supporting a much more complex role of RGCs in vision (Atick & Redlich, 1992). Why do the authors not compare the energy model to a model that captures center-surround responses instead? Do the authors mean to claim that the luminance model captures only the pooling aspects of an RGC model? This is particularly confusing as Figures 6 and 9 show the luminance and energy models for original vs synth aligning with the scaling of Midget and Parasol RGCs, respectively. These claims should be more clearly stated, and citations included to motivate this. Similarly, with the energy model, the physiological evidence is very loosely connected to the model discussed.

We have removed the bars showing potential scaling values measured by electrophysiology in the primate visual system and attempted to clarify our language around the relationship between these models and physiology. Our metamer models are only loosely connected to the physiology, and we’ve decided in revision not to imply any direct connection between the model parameters and physiological measurements. The models should instead be understood as loosely inspired by physiology, but not as a tool to localize the representation (as was done in the Freeman paper).

The physiological scaling values are still used as the mean of the priors on the critical scaling value for model fitting, as described on pg. 27, starting on line 698.

Prior Work:

While the explorations in this paper clearly have value, it does not present any particularly groundbreaking results, and those reported are consistent with previous literature.The explorations around critical eccentricity measurement have been done for texture models (Figure 11) in multiple papers (Freeman 2011, Wallis, 2019, Balas 2009). In particular, Freeman 20111 demonstrated that simpler models, representing measurements presumed to occur earlier in visual processing need smaller pooling regions to achieve metamerism. This work’s measurements for the simpler models tested here are consistent with those results, though the model details are different. In addition, Brown, 2023 (which is miscited) also used an extended field of view (though not as large as in this work). Both Brown 2023, and Wallis 2019 performed an exploration of the effect of the target image. Also, much of the more recent previous work uses color images, while the author’s exploration is only done for greyscale.

We were pleased to find consistency of our results with previous studies, given the (many) differences in stimuli and experimental conditions (especially viewing angle), while also extending to new results with the luminance model, and the effects of initialization. Note that only one of the previous studies (Freeman and Simoncelli, 2011) used a pooled spectral energy model. Moreover, of the previous studies, only one (Brown et al., 2023) used color images (we have corrected that citation - thanks for catching the error).

Discussion of Prior Work:

The prior work on testing metamerism between original vs. synthesized and synthesized vs. synthesized images is presented in a misleading way. Wallis et al.’s prior work on this should not be a minor remark in the post-experiment discussion. Rather, it was surely a motivation for the experiment. The text should make this clear; a discussion of Wallis et al. should appear at the start of that section. The authors similarly cite much of the most relevant literature in this area as a minor remark at the end of the introduction (P3L72).

The large differences we observed between comparison types (original vs synthesized, compared to synthesized vs synthesized) surprised us. Understanding such difference was not a primary motivation for the work, but it is certainly an important component of our results. In the introduction, we thought it best to lay out the basic logic of the metamer paradigm for foveated vision before mentioning the complications that are introduced in both the Wallis and Brown papers (paragraph beginning p. 3, line 109). Our results confirm and bolster the results of both of those earlier works, which are now discussed more fully in the Introduction (lines 109 and following).

White Noise: The authors make an analogy to the inability of humans to distinguish samples of white noise. It is unclear however that human difficulty distinguishing samples of white noise is a perceptual issue- It could instead perhaps be due to cognitive/memory limitations. If one concentrates on an individual patch one can usually tell apart two samples. Support for these difficulties emerging from perceptual limitations, or a discussion of the possibility of these limitations being more cognitive should be discussed, or a different analogy employed.

We now note the possibility of cognitive limits on pg. 8, starting on line 243, as well as pg. 22, line 571. The ability of observers to distinguish samples of white noise is highly dependent on display conditions. A small patch of noise (i.e., large pixels, not too many) can be distinguished, but a larger patch cannot, especially when presented in the periphery. This is more generally true for textures (as shown in Ziemba and Simoncelli (2021)). Samples of white noise at the resolution used in our study are indistinguishable.

Relatedly, in Figure 14, the authors do not explain why the white noise seeds would be more likely to produce syntheses that end up in different human equivalence classes.

In figure 14, we claim that white noise seeds are more likely to end up in the same human equivalence classes than natural image seeds. The explanation as to why we think this may be the case is now addressed on pg. 19, starting on line 423.

It would be nice to see the effect of pink noise seeds, which mirror the power spectrum of natural images, but do not contain the same structure as natural images - this may address the artifacts noted in Figure 9b.

The lack of pink noise seeds is now addressed on pg. 19, starting on line 429.

Finally, the authors note high-frequency artifacts in Figure 4 & P5L135, that remain after syntheses from the luminance model. They hypothesize that this is due to a lack of constraints on frequencies above that defined by the pooling region size. Could these be addressed with a white noise image seed that is pre-blurred with a low pass filter removing the frequencies above the spatial frequency constrained at the given eccentricity?

The explanation for this is similar to the lack of pink noise seeds in the previous point: the goal of metamer synthesis is model testing, and so for a given model, we want to find model metamers that result in the smallest possible critical scaling value. Taking white noise seed images and blurring them will almost certainly remove the high frequencies visible in luminance metamers in figure 4 and thus result in a larger critical scaling value, as the reviewer points out. However, the logic of the experiments requires finding the smallest critical scaling value, and so these model metamers would be uninformative. In an early stage of the project, we did indeed synthesize model metamers using pink noise seeds, and observed that the high frequency artifacts were less prominent.

Schematic of metamerism: Figures 1,2,12, and 13 show a visual schematic of the state space of images, and their relationship to both model and human metamers. This is depicted as a Voronoi diagram, with individual images near the center of each shape, and other images that fall at different locations within the same cell producing the same human visual system response. I felt this conceptualization was helpful. However, implicitly it seems to make a distinction between metamerism and JND (just noticeable difference). I felt this would be better made explicit. In the case of JND, neighboring points, despite having different visual system responses, might not be distinguishable to a human observer.

Thanks for noting this – in general, metamers are subthreshold, and for the purpose of the diagram, we had to discretize the space showing metameric regions (Voronoi regions) around a set of stimuli. We’ve rewritten the captions to explain this better. We address the binary subthreshold nature of the metamer paradigm in the discussion section (pg. 19, line 438).

In these diagrams and throughout the paper, the phrase ’visual stimulus’ rather than ’image’ would improve clarity, because the location of the stimulus in relation to the fovea matters whereas the image can be interpreted as the pixels displayed on the computer.

We agree and have tried to make this change, describing this choice on pg. 3 line 73.

Other

The authors show good reproducibility practices with links to relevant code, datasets, and figures.

Reviewer #1 (Recommendations For The Authors):

In its current form, I found the introduction to be too cursory. I felt that the article would benefit from a clearer motivation for the two models that are considered as the reader is left unclear why these particular models are of special scientific significance. The luminance model is intended to capture some aspects of retinal ganglion cells response characteristics and the spectral energy model is intended to capture some aspects of the primary visual cortex. However, one can easily imagine models that include the pooling of other kinds of features, and it would be helpful to get an idea of why these are not considered. Which aspects of processing in the retina and V1 are being considered and which are being left out, and why? Why not consider representations that capture even higher-order statistical structure than those covered by the spectral energy model (or even semantics)? I think a bit of rewriting with this in mind could improve the introduction.

Along similar lines, I would have appreciated having the logic of the study explained more explicitly and didactically: which overarching research question is being asked, how it is operationalised in the models and experiments, and what are the predictions of the different models. Figures 2 and 3 are certainly helpful, but I felt further explanations would have made it easier for the reader to follow. Throughout, the writing could be improved by a careful re-reading with a view to making it easier to understand. For example, where results are presented, a sentence or two expanding on the implications would be helpful.

I think the authors could also be more explicit about the assumptions they make. While these are obviously (tacitly) included in the description of the models themselves, it would be helpful to state them more openly. To give one example, when introducing the notion of critical scaling, on p.6 the authors state as if it is a self-evident fact that "metamers can be achieved with windows whose size is matched to that of the underlying visual neurons". This presumably is true only under particular conditions, or when specific assumptions about readout from populations of neurons are invoked. It would be good to identify and state such assumptions more directly (this is partly covered in the Discussion section ’The linking proposition underlying the metamer paradigm’, but this should be anticipated or moved earlier in the text).

We agree that our introduction was too cursory and have reworked it. We have also backed off of the direct comparison to physiology and clarified that we chose these two as the simplest possible pooling models. We have also added sentences at the end of each result section attempting to summarize the implication (before discussing them fully in the discussion). Hopefully the logic and assumptions are now clearer.

There are also some findings that warrant a more extensive discussion. For example, what is the broader implication of the finding that original vs. synthesised and synthesised vs. synthesised comparisons exhibit very different scaling values? Does this tell us something about internal visual representations, or is it simply capturing something about the stimuli?

We believe this difference is a result of the stimuli that are used in the experiment and thus the synthesis procedure itself, which interacts with the model’s pooled image feature. We have attempted to update the relevant figures and discussions to clarify this, in the sections starting on pg 17 line 396 and pg. 19 line 417.

At some points in the paper, a third model (’texture model’) creeps into the discussion, without much explanation. I assume that this refers to models that consider joint (rather than marginal) statistics of wavelet responses, as in the famous Portilla & Simoncelli texture model. However, it would be helpful to the reader if the authors could explain this.

Addressed on pg. 3, starting on line 94.

Minor corrections.

Caption of Figure 3: ’top’ and ’bottom’ should be ’left’ and ’right’

Line 177: ’smallest tested scaling values tested’. Remove one instance of ’tested’

Line 212: ’the images-specific psychometric functions’ -> ’image-specific’

Line 215: ’cloud-like pink noise’. It’s not literally pink noise, so I would drop this.

Line 236: ’Importantly, these results cannot be predicted from the model, which gives no specific insight as to why some pairs are more discriminable than others’. The authors should specify what we do learn from the model if it fails to provide insight into why some image pairs are more discriminable than others.

Figure 9: it might be helpful to include small insets with the ’highway’ and ’tiles’ source images to aid the reader in understanding how the images in 9B were generated.

Table 1 placement should be after it is first referred to on line 258.

In the Discussion section "Why does critical scaling depend on the comparison being performed", it would be helpful to consider the case where the two model metamers *are* distinguishable from each other even though each is indistinguishable from the target image. I would assume that this is possible (e.g., if the target image is at the midpoint between the two model images in image space and each of the stimuli is just below 1 JND away from the target). Or is this not possible for some reason?

Regarding line 236: this specific line has been removed, and the discussion about this issue has all been consolidated in the final section of the discussion, starting on pg. 19 line 438.

Regarding the final comment: this is addressed in the paragraph starting on pg. 16 line 386. To expand upon that: the situation laid out by the reviewer is not possible in our conceptualization, in which metamerism is transitive and image discriminability is binary. In order to investigate situations like the one laid out by the reviewer, one needs models whose representations have metric properties, i.e., which allow you to measure and reason about perceptual distance, which we refer to in the paragraph starting on pg. 20 line 460. We also note that this situation has not been observed in this or any other pooling model metamer study that we are aware of. All other minor changes have been addressed.

Reviewer #2 (Recommendations For The Authors):

Original image T should be marked in the Voronoi diagrams.

Brown et al is miscited as 2021 should be ACM Transactions on Applied Perception 2023.

Figure 3 caption: models are left and right, not top and bottom.

Thanks, all of the above have been addressed.

References

BrownReral Encoding, in the Human Visual System. ACM Transactions on Applied Perception. 2023 Jan; 20(1):1–22.http://dx.doi.org/10.1145/356460, Dutell V, Walter B, Rosenholtz R, Shirley P, McGuire M, Luebke D. Efficient Dataflow Modeling of Periph-5, doi: 10.1145/3564605.

Freeman Jdoi: 10.1038/nn.2889, Simoncelli EP. Metamers of the ventral stream. Nature Neuroscience. 2011 aug; 14(9):1195–1201..

Ziemba CMnications. 2021 jul; 12(1)., Simoncelli EP. Opposing Effects of Selectivity and Invariance in Peripheral Vision. Nature Commu-https://doi.org/10.1038/s41467-021-24880-5, doi: 10.1038/s41467-021-24880-5.

eLife Assessment

This study presents a valuable finding relating to how the state of arousal is represented within the superior colliculus, a principal visuo-oculomotor structure. The main conclusion that the representation of arousal is segregated, and thus influences visual activity but not motor output, is incompletely supported by the evidence, but could be stronger if a specific concern relating to an alternative explanation for the dichotomy was addressed. The work will be of interest to sensory, motor, and cognitive neuroscientists.

Reviewer #1 (Public review):

Summary:

Johnston and Smith used linear electrode arrays to record from small populations of neurons in the superior colliculus (SC) of monkeys performing a memory-guided saccade (MGS) task. Dimensionality reduction (PCA) was used to reveal low-dimensional subspaces of population activity reflecting the slow drift of neuronal signals during the delay period across a recording session (similar to what they reported for parts of cortex: Cowley et al., 2020). This SC drift was correlated with a similar slow-drift subspace recorded from the prefrontal cortex, and both slow-drift subspaces tended to be associated with changes in arousal (pupil size). These relationships were driven primarily by neurons in superficial layers of the SC, where saccade sensitivity/selectivity is typically reduced. Accordingly, delay-period modulations of both spiking activity and pupil size were independent of saccade-related activity, which was most prevalent in deeper layers of the SC. The authors suggest that these findings provide evidence of a separation of arousal- and motor-related signals. The analysis techniques expand upon the group's previous work and provides useful insight into the power of large-scale neural recordings paired with dimensionality reduction. This is particularly important with the advent of recording technologies which allow for the measurement of spiking activity across hundreds of neurons simultaneously. Together, these results provide a useful framework for comparing how different populations encode signals related to cognition, arousal, and motor output in potentially different subspaces.

Comments on revised manuscript:

The authors have done a very good job of responding to all of the reviewers' concerns.

Reviewer #2 (Public review):

Summary:

Neurons in motor-related areas have increasingly shown to carry also other, non-motoric signals. This creates a problem of avoidance of interference between the motor and non-motor-related signals. This is a significant problem that likely affects many brain areas. The specific example studied here is interference between saccade-related activity and slow-changing arousal signals in the superior colliculus. The authors identify neuronal activity related to saccades and arousal. Identifying saccade-related activity is straightforward, but arousal-related activity is harder to identify. The authors first identify a potential neuronal correlate of arousal using PCA to identifying a component in the population activity corresponding to slow drift over the recording session. Next, they link this component to arousal by showing that the component is present across different brain areas (SC and PFC), and that it is correlated with pupil size, an external marker of arousal. Having identified an arousal-related component in SC, the authors show next that SC neurons with strong motor-related activity are less strongly affected by this arousal component (both SC and PFC). Lastly, they show that SC population activity pattern related to saccades and pupil size form orthogonal subspaces in the SC population.

Strengths:

A great strength of this research is the clear description of the problem, its relationship with the performed analysis and the interpretation of the results. the paper is very well written and easy to follow. An additional strength is the use of fairly sophisticated analysis using population activity.

Weaknesses:

(1) The greatest weakness in the present research is the fact that arousal is a functionally less important non-motoric variable. The authors themself introduce the problem with a discussion of attention, which is without any doubt the most important cognitive process that needs to be functionally isolated from oculomotor processes. Given this introduction, one cannot help but wonder, why the authors did not design an experiment, in which spatial attention and oculomotor control are differentiated. Absent such an experiment, the authors should spend more time on explaining the importance of arousal and how it could interfere with oculomotor behavior.

(2) In this context, it is particularly puzzling that one actually would expect effects of arousal on oculomotor behavior. Specifically, saccade reaction time, accuracy, and speed could be influenced by arousal. The authors should include an analysis of such effects. They should also discuss the absence or presence of such effects and how they affect their other results.

(3) The authors use the analysis shown in Figure 6D to argue that across recording sessions the activity components capturing variance in pupil size and saccade tuning are uncorrelated. however, the distribution (green) seems to be non-uniform with a peak at very low and very high correlation specifically. The authors should test if such an interpretation is correct. If yes, where are the low and high correlations respectively? Are there potentially two functional areas in SC?

Comments on revised manuscript:

I remain somewhat concerned that the authors jump immediately into an analysis of the 'arousal-related' effects on SC activity. Before that, I would like to see a more detailed discussion justifying the use pupil size alone (i.e., w/o other indicators such as RT) as indicative of fluctuations in general arousal that are causal to concomitant changes in SC activity. Instead, in its current form, the authors find changes in SC activity and describe them immediately as 'arousal-related'.

Other than this conceptual issue, I do not have major problems with the analysis per se.

Reviewer #3 (Public review):

Summary:

This study looked at slow changes in neuronal activity (on the order of minutes to hours) in the superior colliculus (SC) and prefrontal cortex (PFC) of two monkeys. They found that SC activity shows slow drift in neuronal activity like in the cortex. They then computed a motor index in SC neurons. By definition, this index is low if the neuron has stronger visual responses than motor response, and it is low if the neuron has weaker visual responses and stronger motor responses. The authors found that the slow drift in neuronal activity was more prevalent in the low motor index SC neurons and less prevalent in the high motor index neurons. In addition, the authors measured pupil diameter and found it to correlate with slow drifts in neuronal activity, but only in the neurons with lower motor index of the SC. They concluded that arousal signals affecting slow drifts in neuronal modulations are brain-wide. They also concluded that these signals are not present in the deepest SC layers, and they interpreted this to mean that this minimizes the impact of arousal on unwanted eye movements.

Strengths:

The paper is clear and well-written.

Showing slow drifts in the SC activity is important to demonstrate that cortical slow drifts could be brain-wide.

Weaknesses:

The authors find that the SC cells with the low motor index are modulated by pupil diameter. However, this could be completely independent of an "arousal signal". These cells have substantial visual sensitivity. If the pupil diameter changes, then their activity should be influenced since the monkey is watching a luminous display. So, in this regard, the fact that they do not see "an arousal signal" in the most motor neurons (through the pupil diameter analyses) is not evidence that the arousal signal is filtered out from the motor neurons. It could simply be that these neurons simply do not get affected by the pupil diameter because they do not have visual sensitivity. So, even with the pupil data, it is still a bit tricky for me to interpret that arousal signals are excluded from the "output layers" of the SC.

Of course, the general conclusion is that the motor neurons will not have the arousal signal. It's just the interpretation that is different in the sense that the lack of the arousal signal is due to a lack of visual sensitivity in the motor neurons.

I think that it is important to consider the alternative caveat of different amounts of light entering the system. Changes in light level caused by pupil diameter variations can be quite large. Please also note that I do not mean the luminance transient associated with the target onset. I mean the luminance of the gray display. it is a source of light. if the pupil diameter changes, then the amount of light entering to the visually sensitive neurons also changes.

Comments on revised manuscript:

The authors have addressed my first primary comment. For the light comment, I'm still not sure they addressed it. At the very least, they should explicitly state the possibility that the amount of light entering from the gray background can matter greatly, and it is not resolved by simply changing the analysis interval to the baseline pre-stimulus epoch. I provide more clear details below:

In line 194 of the redlined version of the article (in the Introduction), the citation to Baumann et al., PNAS, 2023 is missing near the citation of Jagadisan and Gandhi, 2022. Besides replicating Jagadisan and Gandhi, 2022, this other study actually showed that the subspaces for the visual and motor epochs are orthogonal to each other

Line 683 (and around) of the redlined version of the article (in the Results): I'm very confused here. When I mentioned visual modulation by changed pupil diameter, I did not mean the transient changes associated with the brief onset of the cue in the memory-guided saccade task. I meant the gray background of the display itself. This is a strong source of light. If the pupil diameter changes across trials, then the amount of light entering the eye also changes from the gray background. Thus, visually-responsive neurons will have different amount of light driving them. This will also happen in the baseline interval containing only a fixation spot. The arguments made by the authors here do not address this point at all. So, please modify the text to explicitly state the possibility that the global luminance of the display (as filtered by the pupil diameter) alters the amount of light driving the visually-responsive neurons and could contribute to the higher effects seen in the more visual neurons.

The figures (everywhere, including the responses to reviewers) are very low resolution and all equations in methods are missing.

I'm very confused by Fig. 2 - supplement 2. Panel B shows a firing rate burst aligned to *microsaccade* onset. Does that mean you were in the foveal SC? i.e. how can neurons have a motor burst to the target of the memory-guided saccade and also for microsaccades? And which microsaccade directions caused such a burst? And what does it mean to compute the motor index and spike count for microsaccades in panel C? if you were in the proper SC location for the saccade target, then shouldn't you *not* get any microsaccade-related burst at all? This is very confusing to me and needs to be clarified

Author response:

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

Reviewer #1 (Public Review):

(1) The authors make fairly strong claims that "arousal-related fluctuations are isolated from neurons in the deep layers of the SC" (emphasis added). This conclusion is based on comparisons between a "slow drift axis", a low-dimensional representation of neuronal drift, and other measures of arousal (Figures 2C, 3) and motor output sensitivity (Figures 2B, 3B). However, the metrics used to compare the slow-drift axis and motor activity were computed during separate task epochs: the delay period (600-1100 ms) and a perisaccade epoch (25 ms before and after saccade initiation), respectively. As the authors reference, deep-layer SC neurons are typically active only around the time of a saccade. Therefore, it is not clear if the lack of arousal-related modulations reported for deep-layer SC neurons is because those neurons are truly insensitive to those modulations, or if the modulations were not apparent because they were assessed in an epoch in which the neurons were not active. A potentially more valuable comparison would be to calculate a slow-drift axis aligned to saccade onset. 

The reviewer makes an important point that the calculation of an axis can depend critically on the time window of neuronal response. We find when considering this that the slow drift axis is less sensitive to this issue because it is calculated on time-averaged activity over multiple trials. In previous work we found that slow drift calculated on the stimulus evoked response in V4 was very well aligned to slow drift calculated on pre-stimulus spontaneous activity (Cowley et al, Neuron, 2020, Supplemental Figure 3A and 3B). To address this issue in the present data, we compared the axis computed for an example session for neural activity during the delay period and neural activity aligned to saccade onset. As shown new Figure 2 – figure supplement 1 in the revised manuscript, we found a similar lack of arousal-related modulations for deep-layer SC neurons when slow drift was computed using the saccade epoch (25ms before to 25ms after the onset of the saccade). Figure 2 – figure supplement 1A shows loadings for the SC slow drift axis when it was computed using spiking responses during the delay period (as in the main manuscript analysis). In contrast, Figure 2 – figure supplement 1B shows loadings from the same session when the SC slow drift axis was computed using spiking responses during the saccade epoch. The plots are highly similar and in both cases the loadings were weaker for neurons recorded from channels at the bottom of the probe which have a higher motor index. Finally, we found that projections onto the SC slow drift axis for this session were strongly correlated when the slow drift axis was computed using spiking responses during the delay period and the saccade epoch (r = 0.66, p < 0.001, Figure 1C). Taken together, these results suggest that arousal-related modulations are less evident in deep-layer SC neurons irrespective of whether slow drift was computed during the delay or saccade epoch (see also Public Reviews, Reviewer 1, Point 2).

(2) More generally, arousal-related signals may persist throughout multiple different epochs of the task. It would be worthwhile to determine whether similar "slow-drift" dynamics are observed for baseline, sensory-evoked, and saccade-related activity. Although it may not be possible to examine pupil responses during a saccade, there may be systematic relationships between baseline and evoked responses. 

Similar to the point above, slow drift dynamics tend to be similar across different response epochs because they are averaged across many trials and seem to tap into responsivity trends that are robust across epochs. As shown in Author response image 1 below, and the Figure 2 – figure supplement 1 in the revised manuscript, similar dynamics were observed when the SC slow drift axis was computed using spiking responses during the baseline, delay, visual and saccade epochs. We did not investigate differences between baseline and evoked pupil responses in the current paper. However, these effects were characterized in one of our previous papers that focused exclusively on the relationship between slow drift and eye-related metrics (Johnston et al., 2022, Cereb. Cortex, Figure 6). In this previous work, we found a negative correlation between baseline and evoked pupil size. Both variables were significantly correlated with slow drift, the only difference being the sign of the correlation.

Author response image 1.

(A-C) Dynamics of slow drift for three example sessions when the SC slow drift axis was computed using spiking responses during the baseline, delay, visual and saccade epochs. Baseline = 100ms before the onset of the target stimulus; Delay = 600 to 1100ms after the offset of the target stimulus; Stim = 25ms to 125ms after the onset of the target stimulus; Sac = 25ms before to 25ms after the onset of the saccade.

Johnston R, Snyder AC, Khanna SB, Issar D, Smith MA (2022) The eyes reflect an internal cognitive state hidden in the population activity of cortical neurons. Cereb Cortex 32:3331–3346.

(3) The relationships between changes in SC activity and pupil size are quite small (Figures 2C & 5C). Although the distribution across sessions (Figure 2C) is greater than chance, they are nearly 1/4 of the size compared to the PFC-SC axis comparisons. Likewise, the distribution of r2 values relating pupil size and spiking activity directly (Figure 5) is quite low. We remain skeptical that these drifts are truly due to arousal and cannot be accounted for by other factors. For example, does the relationship persist if accounting for a very simple, monotonic (e.g., linear) drift in pupil size and overall firing rate over the course of an individual session? 

Firstly, it is important to note that the strength of the relationship between projections onto the SC slow drift axis and pupil size (r² = 0.06) is within the range reported by Joshi et al. (2016, Neuron, Figure 3). They investigated the median variance explained between the spiking responses of individual SC neurons and pupil size and found it to be approximately 0.02 across sessions. Secondly, our statistical approach of testing the actual distribution of r² values against a shuffled distribution was specifically designed to rule out the possibility that the relationship between SC spiking responses and pupil size occurred due to linear drifts. The shuffled distribution in Figure 2C of the main manuscript represents the variance that can be explained by one session’s slow drift correlated with another session’s pupil, which would contain effects that occurred due to linear drifts alone. That the actual proportion of variance explained was significantly greater than this distribution suggests that the relationship between projections onto the SC slow drift axis and pupil size reflects changes in arousal rather than other factors related to linear drifts.

Joshi S, Li Y, Kalwani RM, Gold JI (2016) Relationships between Pupil Diameter and Neuronal Activity in the Locus Coeruleus, Colliculi, and Cingulate Cortex. Neuron 89:221–234.

(4) It is not clear how the final analysis (Figure 6) contributes to the authors' conclusions. The authors perform PCA on: (i) residual spiking responses during the delay period binned according to pupil size, and (ii) spiking responses in the saccade epoch binned according to target location (i.e., the saccade tuning curve). The corresponding PCs are the spike-pupil axis and the saccade tuning axis, respectively. Unsurprisingly, the spikepupil axis that captures variance associated with arousal (and removes variance associated with saccade direction) was not correlated with a saccade-tuning axis that captures variance associated with saccade direction and omits arousal. Had these measures been related it would imply a unique association between a neuron's preferred saccade direction and pupil control- which seems unlikely. The separation of these axes thus seems trivial and does not provide evidence of a "mechanism...in the SC to prevent arousal-related signals interfering with the motor output." It remains unknown whether, for example, arousal-related signals may impact trial-by-trial changes in neuronal gain near the time of a saccade, or alter saccade dynamics such as acceleration, precision, and reaction time. 

The reviewer makes a good point, and we agree that more evidence is needed to determine if the separation of the pupil size axis and saccade tuning axis is the mechanism through which cognitive and arousal-related signals can be intermixed in the SC. In the revised manuscript (lines 679-682), we have raised this as a possible explanation that necessitates further study rather than stating definitively that it is the exact mechanism through which these signals are kept separate. Our analysis here is similar to the one from Smoulder et al (2024, Neuron, Fig. 2F), in which the interactions between reward signals and target tuning in M1 were examined (and found to be orthogonal). While we agree with the reviewer that it may seem “trivial” for these axes to be orthogonal, it does not have to be so. If, for example, neural tuning curves shifted with changes in pupil size through gain changes that revealed tuning or affected tuning curve shape, there could be projections of the pupil axis onto the target tuning axis. Thus, while we agree with the reviewer that it appears sensible for these two axes to be orthogonal, our result is nonetheless a novel finding. We have edited the text in our revised manuscript, however, to make sure the nuance of this point is conveyed to the reader.

Smoulder AL, Marino PJ, Oby ER, Snyder SE, Miyata H, Pavlovsky NP, Bishop WE, Yu BM, Chase SM, Batista AP. A neural basis of choking under pressure. Neuron. 2024 Oct 23;112(20):3424-33.

Reviewer #2 (Public Review):

(1) The greatest weakness in the present research is the fact that arousal is a functionally less important non-motoric variable. The authors themselves introduce the problem with a discussion of attention, which is without any doubt the most important cognitive process that needs to be functionally isolated from oculomotor processes. Given this introduction, one cannot help but wonder, why the authors did not design an experiment, in which spatial attention and oculomotor control are differentiated. Absent such an experiment, the authors should spend more time explaining the importance of arousal and how it could interfere with oculomotor behavior. 

Although attention does represent an important cognitive process, we did not design an experiment in which attention and oculomotor control are differentiated because attention does not appear to be related to slow drift. In our first paper that reported on this phenomenon, we investigated the effects of spatial attention on slow fluctuations in neural activity by cueing the monkeys to attend to a stimulus in the left or right visual field in a block-wise manner. Each block lasted ~20 minutes and we found that slow drift did not covary with the timing of cued blocks (see Figure 4A, Cowley et al., 2020, Neuron). Furthermore, there is a large body of work showing that arousal also impacts motor behavior leading to changes in a range of eye-related metrics (e.g., pupil size, microsaccade rate and saccadic reaction time - for review, see Di Stasi et al. 2013, Neurosci. Biobehav. Rev.). We also note that the terms attention and arousal are often used in nonspecific and overlapping ways in the literature, adding to some potential confusion here. Nonetheless, pupil-linked arousal is an important variable that impacts motor performance. This has now been stated clearly in the Introduction of the revised manuscript (lines 108-114) to address the reviewer’s concerns and highlight the importance of studying how precise fixation and eye movements are maintained even in the presence of signals related to ongoing changes in brain state. 

Cowley BR, Snyder AC, Acar K, Williamson RC, Yu BM, Smith MA (2020) Slow Drift of Neural Activity as a Signature of Impulsivity in Macaque Visual and Prefrontal Cortex. Neuron 108:551-567.e8.

(2) In this context, it is particularly puzzling that one actually would expect effects of arousal on oculomotor behavior. Specifically, saccade reaction time, accuracy, and speed could be influenced by arousal. The authors should include an analysis of such effects. They should also discuss the absence or presence of such effects and how they affect their other results. 

As described above, several studies across species have demonstrated that arousal impacts motor behavior e.g., saccade reaction time, saccade velocity and microsaccade rate (for review, see Di Stasi et al. 2013, Neurosci. Biobehav. Rev.). This has been clarified in the Introduction of the revised manuscript to address the reviewer's concerns (lines 108-114). Our prior work (Johnston et al, Cerebral Cortex, 2022) shows that slow drift impacts several types of oculomotor behavior. Overall, these studies highlight the impact of arousal on eye movements as a robust effect, and support the present investigation into arousal and oculomotor control signals. While we agree reaction time, accuracy, and speed all can be influenced by arousal depending on task demands, the present study is focused on the connection between slow fluctuations in neural activity, linked to arousal, and different subpopulations of SC neurons. 

Di Stasi LL, Catena A, Cañas JJ, Macknik SL, Martinez-Conde S (2013) Saccadic velocity as an arousal index in naturalistic tasks. Neurosci Biobehav Rev 37:968–975.

Johnston R, Snyder AC, Khanna SB, Issar D, Smith MA (2022) The eyes reflect an internal cognitive state hidden in the population activity of cortical neurons. Cereb Cortex 32:3331–3346.

(3) The authors use the analysis shown in Figure 6D to argue that across recording sessions the activity components capturing variance in pupil size and saccade tuning are uncorrelated. however, the distribution (green) seems to be non-uniform with a peak at very low and very high correlation specifically. The authors should test if such an interpretation is correct. If yes, where are the low and high correlations respectively? Are there potentially two functional areas in SC? 

We agree with the reviewer that our actual data distribution was non-uniform. We examined individual sessions with high and low variance explained and did not find notable differences. One source of this variation has to do with session length. Longer sessions in principle should have a chance distribution of variance explained closer to zero because they contained more time bins. Given that we had no specific hypothesis for a non-uniform distribution, we have simply displayed the full distribution of values in our figure and the statistical result of a comparison to a shuffled distribution.

Reviewer #3 (Public Review):

(1) However, I am concerned about two main points: First, the authors repeatedly say that the "output" layers of the SC are the ones with the highest motor indices. This might not necessarily be accurate. For example, current thresholds for evoking saccades are lowest in the intermediate layers, and Mohler & Wurtz 1972 suggested that the output of the SC might be in the intermediate layers. Also, even if it were true that the high motor index neurons are the output, they are very few in the authors' data (this is also true in a lot of other labs, where it is less likely to see purely motor neurons in the SC). So, this makes one wonder if the electrode channels were simply too deep and already out of the SC? In other words, it seems important to show distributions of encountered neurons (regardless of the motor index) across depth, in order to better know how to interpret the tails of the distributions in the motor index histogram and in the other panels of Figure Supplement 1. I elaborate more on these points in the detailed comments below. 

The reviewer makes a good point about the efferent signals from SC. It is true that electrical thresholds are often lowest in intermediate layers, though deep layers do project to the oculomotor nuclei (Sparks, 1986; Sparks & Hartwich-Young, 1989) and often intermediate and deep layers are considered to function together to control eye movements (Wurtz & Albano, 1980). As suggested by the reviewer, we have edited the text throughout the manuscript to say that slow drift was less evident in SC neurons with a higher motor index, as well as included the above references and points about the intermediate and deep layers (Lines 73-81). Aside from the question of which layers of the SC function as the “motor output”, the reviewer raises a separate and important question – are our deep recordings still in SC. Here, we can say definitively that they are. We removed neurons if they did not exhibit elevated (above baseline) firing rates during the visual or saccade epochs of the MGS task (see Methods section on “Exclusion criteria”). All included neurons possessed a visual, visuomotor or motor response, consistent with the response properties of neurons in the SC. In addition, we found a number of neurons well above the bottom of the probe with strong motor responses and minimal loadings onto the slow drift axis (see Figure 2 – figure supplement 1A), consistent with the reviewer’s comment that intermediate layer neurons are tuned for movement and play a role in saccade production.

Mohler CW, Wurtz RH. Organization of monkey superior colliculus: intermediate layer cells discharging before eye movements. Journal of neurophysiology. 1976 Jul 1;39(4):722-44.

Sparks DL. Translation of sensory signals into commands for control of saccadic eye movements: role of primate superior colliculus. Physiol Rev. 1986 Jan;66(1):118-71. doi: 10.1152/physrev.1986.66.1.118. PMID: 3511480.

Sparks DL, Hartwich-Young R. The deep layers of the superior colliculus. Reviews of oculomotor research. 1989 Jan 1;3:213-55.

Wurtz RH, Albano JE. Visual-motor function of the primate superior colliculus. Annu Rev Neurosci. 1980;3:189-226. doi: 10.1146/annurev.ne.03.030180.001201. PMID: 6774653.

(2) Second, the authors find that the SC cells with a low motor index are modulated by pupil diameter. However, this could be completely independent of an "arousal signal". These cells have substantial visual responses. If the pupil diameter changes, then their activity should be influenced since the monkey is watching a luminous display. So, in this regard, the fact that they do not see "an arousal signal" in most motor neurons (through the pupil diameter analyses) is not evidence that the arousal signal is filtered out from the motor neurons. It could simply be that these neurons simply do not get affected by the pupil diameter because they do not have visual sensitivity. So, even with the pupil data, it is still a bit tricky for me to interpret that arousal signals are excluded from the "output layers" of the SC. 

The reviewer makes an important point about the SC’s visual responses. Neurons with a low motor index are, conversely, likely to have a stronger visual response index. However, we do not believe that changes in luminance can explain why the correlation between SC spiking response and pupil size is weaker for neurons with a lower motor index. Firstly, the changes in pupil size observed in the current paper and our previous work are slow and occur on a timescale of minutes (Cowley et al., 2020, Neuron) and are correlated with eye movement measures such as reaction time and microsaccade rate (Johnston et al., 2022, Cerebral Cortex). This is in stark contrast to luminance-evoked changes in pupil size that occur on a timescale of less than a second. Secondly, as shown the new Figure 5 – figure supplement 1 in the revised manuscript, very similar results were found when SC spiking responses were correlated with pupil size during the baseline period, when only the fixation point was on the screen. Although the luminance of the small peripheral target stimulus can result in small luminance-evoked changes in pupil size, no changes in luminance occurred during the baseline period which was defined as 100ms before the onset of the target stimulus. In Figure 2 – figure supplement 1 and Author response image 1 above, we show that slow drift is the same whether calculated on the baseline response, delay period, or peri-saccadic epoch. Thus, the measurement of slow drift is insensitive to the precise timing of the selection of both the window for the spiking response and the window for the pupil measurement. If luminance were the explanation for the slow changes in firing observed in visually responsive SC neurons, it would require those neurons to exhibit robust, sustained tuned responses to the small changes in retinal illuminance induced by the relatively small fluctuations in pupil size we observed from minute to minute. We are aware of no reports of such behavior in visually-responsive neurons in SC. We have included these analyses and this reasoning in the revised manuscript on lines 478-495.

Reviewer#1 (Recommendations for the author):

(1) It would be useful to provide line numbers in subsequent manuscripts for reviewers.

Line numbers have been added in the revised version of the manuscript.

(2) Page #6; last sentence: "...even impact processing at the early to mid stages of the visuomotor transformation, without leading to unwanted changes in motor output." I do not believe the authors have provided evidence that arousal levels were not associated with changes in motor output.

As suggested by Reviewer 3 (see Public Reviews, Reviewer 3, Point 2), we have edited the text throughout the manuscript to say that slow drift was less evident in SC neurons with a higher motor index. This sentence in the revised manuscript now reads:

“This provides a potential mechanism through which signals related to cognition and arousal can exist in the SC, and even impact processing at the early to mid stages of the visuomotor transformation, without leading to unwanted changes in SC neurons that are linked to saccade execution.”

(3) Page #8; last paragraph: Although deep-layer SC neurons may not have been obtained during every recording session, a summary of the motor index scores observed along the probe across sessions would be useful to confirm their assumptions. 

See Author response image 2 below which shows the motor index of each recoded SC neuron on the x-axis and session number on the y-axis. The points are colored by to the squared factor loading which represents the variance explained between the response a neuron and the slow drift axis (see Figure 3B of the main manuscript). You can see from this plot that neurons with a stronger component loading (shown in teal to yellow) typically have a lower motor index whereas the opposite is true for neurons with a weaker component loading (shown in dark blue).

Author response image 2.

Scatter plot showing the motor index of each recorded neuron along with the session number in which it was recorded. The points are colored by to the squared factor loading for each neuron along the slow drift axis. Note that loadings above 0.5 (33 data points in total) have been thresholded at 0.5 so that we could effectively use the color range to show all of the slow drift axis loadings.

(4) Page #10; first paragraph: The authors should state the time window of the delay period used, since it may be distinct from the pupil analysis (first 200ms of delay). 

This has been stated in the revised version of the manuscript. The sentence now reads:

“We first asked if arousal-related fluctuations are present in the SC. As in previous studies that recorded from neurons in the cortex (Cowley et al., 2020), we found that the mean spiking responses of individual SC neurons during the delay period (chosen at random on each trial from a uniform distribution spanning 600-1100ms, see Methods) fluctuated over the course of a session while the monkeys performed the MGS task (Figure 2A, left).”

(5) Page #10; second paragraph: Extra period at the end of a sentence: " most variance in the data..". 

Fixed in the revised version of the manuscript.

(6) Page #12: "between projections onto the SC slow drift axis and mean pupil size during the first 200ms of the delay period when a task-related pupil response could be observed." What criteria was used to determine whether a task-related pupil response was observed? 

This was chosen based on the results of a previous study in our lab that used the same memory-guided saccade task to investigate the relationship between slow drift and changes in based and evoked pupil size (see Johnston et al., 2022, Cereb. Cortex, Figure 6B). The period was chosen based on plotting the average pupil size aligned on different trial epochs. As we show in Figure 5-figure supplement 3 above, the pupil interactions with slow drift did not depend on the particular time window of the pupil we chose.  

(7) Page #14; Figure 2A: The axes for the individual channels are strangely floating and quite different from all other figures. Please label the channel in the figure legend that was used as an example of the projected values onto the slow drift axis.

The figure has been changed in the revised version of the manuscript so that the tick mark denoting zero residual spikes per second is on the top layer of each plot. A scale bar was chosen instead of individual axes to reduce clutter in the figure as it was used to demonstrate how slow drift was computed. Residual spiking responses from all neurons were projected on the slow drift axis to generate the scatter plot in the bottom right-hand corner of Figure 2A. There is no single neuron to label.

(8) Page #16: "These results demonstrate that even though arousal-related fluctuations are present in the SC, they are isolated from deep-layer neurons that elicit a strong saccadic response and presumably reside closer to the motor output." In line with our major comments, lack of arousal-related activity during the delay period is meaningless for deep-layer SC neurons that are generally inactive during this time. It does not imply that there is no arousal signal! 

Addressed in Public Reviews, Reviewer 1, Point 1 & 2. We found a similar lack of arousal-related modulations reported for deep-layer SC neurons when slow drift was computed using the saccade epoch (Figure 1 above). In addition, similar dynamics were observed when the SC slow drift axis was computed using spiking responses during the baseline, delay, visual and saccade period (Figure 2).

(9) Page #18: "These findings provide additional support for the hypothesis that arousalrelated fluctuations are isolated from neurons in the deep layers of the SC." The same criticism from above applies.

Addressed in Public Reviews, Reviewer 1, Point 1 & 2.

(10) Page #20; paragraph 3: "Taken together, the findings outlined above..." Would be useful to be more specific when referring to "activity" ; e.g., "...these neurons did not exhibit large fluctuations in delay-period activity over time".

This sentence has been changed in the revised manuscript in light of the reviewer’s comments. It now reads:

“In addition to being more weakly correlated with pupil size, the spiking responses of these neurons did not exhibit large fluctuations over time (Figure 2), and when considering the neuronal population as a whole, explained less variance in the slow drift axis when it was computed using population activity in the SC (Figure 3) and PFC (Figure 4).”

Reviewer #3 (Recommendations for the author):

The paper is clear and well-written. However, I am concerned about two main points: 

(1) First, the authors repeatedly say that the "output" layers of the SC are the ones with the highest motor indices. This might not necessarily be accurate. For example, current thresholds for evoking saccades are lowest in the intermediate layers, and Mohler & Wurtz 1972 suggested that the output of the SC might be in the intermediate layers. Also, even if it were true that the high motor index neurons are the output, they are very few in the authors' data (this is also true in a lot of other labs, where it is less likely to see purely motor neurons in the SC). So, this makes one wonder if the electrode channels were simply too deep and already out of the SC. In other words, it seems important to show distributions of encountered neurons (regardless of motor index) across depth, in order to better know how to interpret the tails of the distributions in the motor index histogram and in the other panels of the figure supplement 1. I elaborate more on these points in the detailed comments below. 

Addressed in Public Reviews, Reviewer 3, Point 1.

(2) Second, the authors find that the SC cells with a low motor index are modulated by pupil diameter. However, this could be completely independent of an "arousal signal". These cells have substantial visual responses. If the pupil diameter changes, then their activity should be influenced since the monkey is watching a luminous display. So, in this regard, the fact that they do not see "an arousal signal" in most motor neurons (through the pupil diameter analyses) is not evidence that the arousal signal is filtered out from the motor neurons. It could simply be that these neurons simply do not get affected by the pupil diameter because they do not have visual sensitivity. So, even with the pupil data, it is still a bit tricky for me to interpret that arousal signals are excluded from the "output layers" of the SC. 

Addressed in Public Reviews, Reviewer 3, Point 2.

(3) I think that a remedy to the first point above is to change the text to make it a bit more descriptive and less interpretive. For example, just say that the slow drifts were less evident among the neurons with high motor index. 

We thank the reviewer for this suggestion (see Public Reviews, Reviewer 3, Point 1).

(4) For the second point, I think that it is important to consider the alternative caveat of different amounts of light entering the system. Changes in light level caused by pupil diameter variations can be quite large. 

We thank the reviewer for this suggestion (see Public Reviews, Reviewer 3, Point 2).

(5) Line 31: I'm a bit underwhelmed by this kind of statement. i.e. we already know that cognitive processes and brain states do alter eye movements, so why is it "critical" that high precision fixation and eye movements are maintained? And, isn't the next sentence already nulling this idea of criticality because it does show that the brain state alters the SC neurons? In fact, cognitive processes are already known to be most prevalent in the intermediate and deep layers of the SC. 

It seems clear that while cognitive state does affect eye movements, it is desirable to have some separation between cognitive state and eye movement control. Covert attention, for instance, is precisely a situation where eye movement control is maintained to avoid overt saccades to the attended stimulus, and yet there are clear indications of attention’s impact on microsaccades and fixation. We stand by our statement that an important goal of vision is to have precise fixation and movements of the eye, and yet at the same time the eyes are subject to numerous influences by cognitive state.

(6) Line 65: it is better to clarify that these are "functional layers" because there are actually more anatomical layers. 

We have edited this sentence in the revised version of the manuscript so that it now reads:

“The role of these projections in the visuomotor transformation depends on the functional layer of the SC in which they terminate”.

(7) Line 73: this makes it sound like only the deepest layers are topographically organized, which is not true. Also, as early as Mohler & Wurtz, 1972, it was suggested that the intermediate layers have the biggest impacts downstream of the SC. This is also consistent with electrical microstimulation current thresholds for evoking saccades from the SC. 

We have addressed the reviewers’ comments about the intermediate layers having the biggest impact downstream of the SC in Public Reviews, Reviewer 3, Point 1. Furthermore, line 73 has been changed in the revised manuscript so that it now reads:

“As is the case for neurons in the superficial and intermediate layers, they [SC motor neurons] form a topographically organized map of visual space (White et al. 2017; Robinson 1972; Katnani and Gandhi 2011)”.  

(8) Line 100: there is an analogous literature regarding the question of why unwanted muscle contractions do not happen. Specifically, in the context of why SC visual bursts do not automatically cause saccades (which is a similar problem to the ones you mention about cognitive signals interfering by generating unwanted eye movements), both Jagadisan & Gandhi, Curr Bio, 2022 and Baumann et al, PNAS, 2023 also showed that SC population activity not only has different temporal structure (Jagadisan & Gandhi) but also occupy different subspaces (Baumann et al) under these two different conditions (visual burst versus saccade burst). This is conceptually similar to the idea that you are mentioning here with respect to arousal. So, it is worth it to mention these studies here and again in the discussion. 

We are grateful to the reviewer for these suggestions and have included text in the Introduction (Lines 125-128) and Discussion (Lines 678-682) of the revised manuscript along with the references cited above.

(9) Line 147: as mentioned above, it is now generally accepted that there are quite a few "pure" motor neurons in the SC. This is consistent with what you find. E.g. Baumann et al., 2023. And, again see Mohler and Wurtz in the 1970's. So, I wonder how useful it is to go too much into this idea of the deeper motor neurons (e.g. the correlations in the other panels of the Figure 1 supplement). 

This is related to the reviewer’s comment that the output of the SC might be in the intermediate layers. This concern has been addressed in Public Reviews, Reviewer 3, Point 1.

(10) Figure 1 should say where the RF was for the shown spike rasters. i.e. were these the same saccade target across trials? And where was that location relative to the RF? It would help also in the text to say whether the saccade was always to the RF center or whether you were randomizing the target location. 

We centered the array of saccade targets using the microstimulation-evoked eye movement for SC (see Methods section “Memory-guided saccade task”) to find the evoked eccentricity, and then used saccade targets with equal spacing of 45 degrees starting at zero (rightward saccade target). We did not do extensive RF mapping beyond this microstimulation centering. In Figure 1, the spike rasters are shown for a target that was visually identified to be within the neuron’s RF based on assessing responses to all 8 target angles. We have added information about this to the figure caption.

(11) Line 218: but were there changes in the eye movement statistics? For example, the slow drift eye movements during fixation? Or even the microsaccades? 

Addressed in Public Reviews, Reviewer 2, Point 2.  

(12) Line 248: shuffling what exactly? I think that more explanation would be needed here. 

Addressed in Public Reviews, Reviewer 1, Point 3.  

(13) Line 263: but isn't this reflecting a sensory transient in the pupil diameter, since the target just disappeared? 

Addressed in Public Reviews, Reviewer 3, Point 2.  

(14) Line 271: I suspect that slow drift eye movements (in between microsaccades) would show higher correlations. Not sure how well you can analyze those with a video-based eye tracker. 

We agree that fixational drift would be a worthwhile metric, but it is not one we have focused on here and to our knowledge does require higher precision tracking. 

(15) Line 286: again, see above about similar demonstrations with respect to the visual and motor burst intervals, which clearly cause the same problem (even stronger) as the one studied here. 

See reply, including Figure 2.

(16) Line 330: again, I'm not sure deeper necessarily automatically means closer to the output. For example, current thresholds for evoked saccades grow higher as you go deeper. Maybe the authors can ask their colleague Neeraj Gandhi about this point specifically, just to be safe. Maybe the safest would be to remain descriptive about the data, and just say something like: arousal-related fluctuations were absent in our deepest recorded sites. 

Addressed in Public Reviews, Reviewer 3, Point 1.

(17) Line 332: likewise, statements like this one here would be qualified if the output was the intermediate layers......anyway if I understand what I read so far in the paper, the signal will be anyway orthogonal to the motor burst population subspace. So, maybe there's no need to emphasize that it goes away in the very deepest layers. 

See reply above, Public Reviews, Reviewer 1, Point 4.

(18) Figure 3A: related to the above, I think one issue could be that the deeper contacts might already be out of the SC. Maybe some cell count distribution from each channel should help in this regard. i.e. were you finding way fewer saccade-related neurons in the deepest channels (even though the few that you found were with high motor index)? If so, then wouldn't this just mean that the channel was too deep? I think there needs to be an analysis like this, to convince readers that the channels were still in the SC. Ideally, electrical stimulation current thresholds for evoking saccades at different depths would be tested, but I understand that this can be difficult at this stage. 

Addressed in Public Reviews, Reviewer 3, Point 1.

(19) I keep repeating this because in general, cognitive effects are stronger in the intermediate/deeper layers than in the superficial layers. If these interfere with eye movements like arousal, then why should arousal be different?

Few studies have investigated the effects of attention on “pure” movement SC neurons that only discharge during a saccade. One study, which we cited in Introduction (Ignashchenkova et al., 2004, Nat. Neurosci.), found significant differences in spiking responses between trials with and without attentional cueing for visual and visuomotor neurons. No significant difference was found for motor neurons, consistent with our hypothesis that signals related to cognition and arousal are kept separate from saccade-related signals in the SC.

(20) The problem with Figure 5 and its related text is that the neurons with low motor index are additionally visual. So, of course, they can be modulated if the pupil diameter changes!

Addressed in Public Reviews, Reviewer 3, Point 2.  

(21) I had a hard time understanding Figure 6. 

See reply above, Public Reviews, Reviewer 1, Point 4.

(22) Line 586: these cells have more visual responses and will be affected by the amount of light entering the eye. 

Addressed in Public Reviews, Reviewer 3, Point 2.

33:55 "They torture Dominika so that she confesses she is a traitor. But you can't kill the Freedom(TM) in her! Did Russia buy out the Guantanamo franchise? For tortures by hard rock? I like that USA in their propaganda assigns to its enemies something that USA has been guilty of themselves. Soviets suddenly drop nuclear bombs on a peaceful city, use chemical weaponry, and suddenly, hard rock tortures."<br /> haha, yeah, every single time... almost as if psychological projection was some natural instinct...<br /> see also: chauvinism, group narcissm, collectivism, hivemind, meme: "Our Blessed Homeland versus Their Barbarous Wastes"

Roots are in capitals, and are not words in use at all, but serve as an elucidation of the words grouped together and a connection between them.

J.R.R. Tolkien's note in the Qenya Lexicon[1]

Sauron new

knew that he had been wrong - not everyone would want to use the ring for their own power and Glory

yes Frodo succumbed at the very very end but - he and Sam made it that far and - fate or Providence or the intervention of Uru? himself did the rest

some people are capable of selfless and purely good acts

it wasn't just just Sauron who fell - it was his entire worldview

hope and love and care and friendship - can triumph over evil - however powerful it may seem at the time

The Lord of the Rings from Sauron's perspective

In Deep Geek 907K subscribers

AI will take job opportunities from musicians, who already struggle to make a living.

I agree, even though commercial music isn't the same as music created by artists, outsourcing it to AI removes career outlets for musicians.

How much money is made in the AI music industry? I will research this next.

This article is 5 years old. I am reading it from today's lens, knowing that AI music technology has advanced far beyond where it was then, making these issues even more prevalent now.

In the AI music business, music is a profit; musicians contribute to the data, and can get compensation for their efforts through redistribution.

Allocating a portion of profits for public use redistributes wealth, which is also good for the economy.

This solved the royalty split issue of who should get credit; the solution was to redistribute the money to the public by helping musicians in need.

AI music generation works the same way as randomly generated melodies; it randomly generates a song based on its training data, which can result in segments that emulate existing songs.

There is a difference between blatantly stealing a melody versus a statistically likely repetition. If you're only looking at three notes of a melody, you will find many songs with those same three notes in the same sequence.

The music industry thrives on recycled ideas; a new idea fuels a new genre, and samples are passed around dozens of times.

There is a line of what can and can't be flagged as a copyright infringement; an identical chord progression can't be sued for, but a sample without permission can.

Meaning some aspects of genres are not able to be copyrighted, things such as common genre drum beats, and chord progressions cannot be copyrighted.

It would be very hard to keep track of the amount of stake both people and machines had in the creation of something to determine royalty splits.

An answer to "Is any music safe from data harvesting?"

This is a fairer way to do this, as now the data isn't from somewhere they don't have permission to use.

This is likely because they have access to data that they dont have direct permission to use.

Should AI products be stripped of all ownership and become public property?

How will this be resolved?

This is an important detail when answering the question of how much each part influences the creation. AIs aren't like humans; they don't need rest.

In AI music creation, the question that needs to be answered is what is the power balance, how much of the creation process is influenced by a users prompt, the data from artists, the process the machine went through to create the song, and the programmers who made the AI music generator.?

Is it ethical to pay a machine?

Is any music safe from data harvesting? There appears to be no safeguard against having your music being used in training data.

That is how these types of services stay afloat, by profiting off your data, and the data it is trained on

This means the business avoids copyright responsibility, as the user is the one who actually generates the music.

So, MIDI websites have significantly affected the music industry, which doesn't exactly answer the research question, but is adjacent. Also I just checked out BitMIdi, it was really weird, it has a ton of instrumenetal versions of songs, basically the elevator music versions of songs. It's really wierd, its versions of songs with chip tune drums, midi saxohpone, and midi strings.

This is a really cool idea.

Wow, i had no idea something like this existed that long ago, I want learn more about how it actually functioned.

I had no idea that generative AI was around 10 years ago; I thought there was only narrow AI with tools such as Siri and Grammarly. This opens my eyes to the hidden landscape of AI in the past decades. The truth is, AI has been around for many decades, and looks very different now than it did before.

the goal of the research in this article is to find a more erthical split in profits among AI models, the user, and the artists that are part of training data.

This means that AI models are trained on music they don't have ownership over, producing music that people profit off of, created from material that was protected under copyright.

There is a discourse in who should profit off of AI generated music.

oracle price 只是 mark_price 的一个 component，还有什么其他因素呢

Her body language says it all.

She's obviously struggling with this decision she is unsure of what will happen.

That's something a person would say who obviously isn't the one going through a procedure not sure if that's comforting or more infuriating.

I found myself intrigued that the film did not end with a happy ending nor a very dark ending either. There was not exactly any clear "good guy" in this film for the acceptation of Brutus who called a "noble man" multiple times in the film. There are many aspects of this film that wrestle with notions of loyalty, honor, and patriotism.

I have to agree with professor Ko that good critique is a conversation over a single opinion. I've been in situations such as group project in my other INFO classes where people can simply say that that looks good or "I think you should fix that small..." without having proper reasoning. ALso the article mention the hamburger rule as it's not just about being nice, but about giving feedback that will help people or project grow. This reminded me that giving critical feedback is important and that also helping the other grow, Personally, I am very much open to critical feedback as long as the reasoning is good!

The authors highlight that the relationship between family income and children’s academic achievement has strengthened over recent decades. This growing “income achievement gap” replaces the older concern about the “race gap.” It shows that economic inequality is now the main barrier to educational equity.

the authors describe schools as the key mechanism through which equal opportunity should be achieved. Education is the “engine” of the American Dream—but it’s also where inequalities are reproduced. This introduces the book’s main theme: the conflict between democratic ideals and educational inequality.

Mark Twain Wrote the First Book Ever Written With a Typewriter<br /> by [[Josh Jones]] in Open Culture<br /> accessed on 2025-10-13T23:01:35