A neck-like vertebral motion in fish

Tetrapods use their neck to move the head three-dimensionally, relative to the body and limbs. Fish lack this anatomical neck, yet during feeding many species elevate (dorsally rotate) the head relative to the body. Cranial elevation is hypothesized to result from the craniovertebral and cranial-most intervertebral joints acting as a neck, by dorsally rotating (extending). However, this has never been tested due to the difficulty of visualizing and measuring vertebral motion in vivo. I used X-ray reconstruction of moving morphology to measure three-dimensional vertebral kinematics in rainbow trout (Oncorhynchus mykiss) and Commerson's frogfish (Antennarius commerson) during feeding. Despite dramatically different morphologies, in both species dorsoventral rotations extended far beyond the craniovertebral and cranial intervertebral joints. Trout combine small (most less than 3°) dorsal rotations over up to a third of their intervertebral joints to elevate the neurocranium. Frogfish use extremely large (often 20–30°) rotations of the craniovertebral and first intervertebral joint, but smaller rotations occurred across two-thirds of the vertebral column during cranial elevation. Unlike tetrapods, fish rotate large regions of the vertebral column to rotate the head. This suggests both cranial and more caudal vertebrae should be considered to understand how non-tetrapods control motion at the head–body interface.

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Do you have any ethical concerns with this paper? No
Comments to the Author I commend the author on starting what I hope will be more study of the functional anatomy of the anterior vertebrae in fishes. This region of the vertebral column is highly variable across fishes and thus likely plays different roles depending on the group. This paper uses a sophisticated technique to analyze the movement of the anterior vertebrae during feeding in 2 quite distinct fish demonstrating that there are d-v intervertebral movements during feeding.
Lines 37-38: While the majority of actinopterygian fish have a connection between the pectoral girdle and the school, this is not true for all actinopterygian fishes or for any cartilaginous fishes. It would be good to include some nuance here given this anatomical diversity.
Lines 93-94: Were only successful strikes included? Also, how often did strikes include cranial elevation less than 5 degrees? Is there a cranial elevation that did not coincide with vertebral movement? I am trying to get an understanding of how often the vertebral column plays in feeding in these two animals.
The author sets up how fish don't have an anatomical neck due to the connection between the pectoral girdle and skull. However, much of the vertebral movement appears to be posterior to this location. While the author does allude to the functional neck movements being a significant part of the anterior vertebral column, this isn't brought into context with the placement of the pectoral girdle. Would you expect more intervertebral movement in species with no connection of the pectoral girdle to the skull?
It would be great for the author to discuss how the movements of the vertebrae are possibly being controlled given that the myomeres responsible for moving these vertebrae span multiple segments. What does the muscular anatomy look like in this region of the body?
My main criticisms is about the broader framing of the study: the fish vs tetrapods comparison is not clear to me. This concerns the description of the observed motions to be neck-like or the fish's vertebral column to have a functional neck as in tetrapods. As I understood, any vertebra performing some degree of rotation in the sagittal plane during the act of suction feeding are interpreted to be perform a neck-like function. What are the arguments for this view? I'm missing this information, as the function of a neck during feeding in tetrapods has not been defined properly in the text. In terrestrial lower vertebrates like lizards, the neck allows the head and jaws to be aimed and moved towards the food in 3D involving both pitch, yaw, and roll, while the trunk remains stationary (e.g. studies by S. Montuelle). If this would be the reference for neck-like function in a tetrapod, I see more differences than similarities with the current results. Namely, the vertebral column in these fish moves in response to a short blast of high-power muscle activity during the strike at the food, while the lizards manage to stably hold the neck into these 3D postures when approaching the food. So at least the temporal and dynamic aspects of these motions do not seem 'neck-like' with respect to lizards. I fully agree that the vertebral column has an important role for head movement during suction feeding. Yet, the author concludes that the vertebral column translates and positions the head, but did not consider the vertebral column to move in response to the dynamics of the head.
Especially for the interpretation of the data of Antennarius, this is important. This animal is experiencing a strong external force in the form of suction propulsion. This forward pulling of the head may cause the straightening of the curves spine. Additionally, the clockwise torque on the head will inevitably push down on the first vertebra, an effect that has also been noted in seahorses (ref 29 in the manuscript; and note examples like the fused four cranialmost vertebra of the powerful head rotator Fistularia seems a response to avoid a collapse of this vertebral region). With this in mind, I'm unsure whether claiming that the data show that the vertebra are translating and positioning the head is appropriate. In other words, I'm afraid that readers will interpret this as an active process driven entirely from within the vertebral musculoskeletal system (statements such as line 59: 'vertebral column producing neck-like motions'), as during slow neck movement in humans, while this is not the case.
Line 27: final sentence -has been written quite a few times now for the muscles surrounding these vertebra and the pectoral girdle. Are you sure you want to repeat this once more, claiming a 'recast' of our scientific view to this dual function? (3) Line 37: 'pectoral girdle attaches directly to the cranium'. There may be some exceptions, like eels. How was it for the shark in your previous publication in Proc. R. Soc B? (4) Line 126 and other places in the manuscript: 'dorsoventral rotations'. As 'dorsovertral' is a lineal axis, this may be less appropriate than 'dorsoventral plane rotations' or 'sagittal plane rotations'. Your manuscript has now been peer reviewed and the reviews have been assessed by an Associate Editor. Based on my own read of your manuscript as well as the assessments of the reviewers and the AE, I find your article to be of potential interest for Proceedings B. However, I agree with the reviewers that there are two major issues that must be addressed prior to further consideration. First, and most importantly, whereas the results are impressive, your study's impact relies on the comparison between the tetrapod neck and the vertebral movement in the fishes. Right now this is not entirely clear, and needs to be better articulated to ensure that the reader understands the impact of your findings. Second, as both reviewers also point out, there needs to be some discussion how neck movement is controlled. Both reviewers' comments and those of the AE are included at the end of this email for your reference.
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Best wishes, Dr Sarah Brosnan Editor, Proceedings B mailto: proceedingsb@royalsociety.org Associate Editor Comments to Author: Both reviewers give the manuscript high marks in general and agree that the data are very valuable, but they raise concerns about the framing and context provided. Most critically, Reviewer 2 questions the functional comparisons between fish and tetrapod necks. What is meant by "neck-like function" should be better defined. In addition, both reviewers request a better explanation of how the head movements are being produced and controlled: by muscular activity? By external forces? Expanding the discussion to address the musculoskeletal anatomy and biomechanical environment of these fish in detail would help to provide a basis for future research in this area.
Reviewer(s)' Comments to Author: Referee: 1 Comments to the Author(s) I commend the author on starting what I hope will be more study of the functional anatomy of the anterior vertebrae in fishes. This region of the vertebral column is highly variable across fishes and thus likely plays different roles depending on the group. This paper uses a sophisticated technique to analyze the movement of the anterior vertebrae during feeding in 2 quite distinct fish demonstrating that there are d-v intervertebral movements during feeding.
Lines 37-38: While the majority of actinopterygian fish have a connection between the pectoral girdle and the school, this is not true for all actinopterygian fishes or for any cartilaginous fishes. It would be good to include some nuance here given this anatomical diversity.
Lines 93-94: Were only successful strikes included? Also, how often did strikes include cranial elevation less than 5 degrees? Is there a cranial elevation that did not coincide with vertebral movement? I am trying to get an understanding of how often the vertebral column plays in feeding in these two animals.
The author sets up how fish don't have an anatomical neck due to the connection between the pectoral girdle and skull. However, much of the vertebral movement appears to be posterior to this location. While the author does allude to the functional neck movements being a significant part of the anterior vertebral column, this isn't brought into context with the placement of the pectoral girdle. Would you expect more intervertebral movement in species with no connection of the pectoral girdle to the skull? It would be great for the author to discuss how the movements of the vertebrae are possibly being controlled given that the myomeres responsible for moving these vertebrae span multiple segments. What does the muscular anatomy look like in this region of the body?
Referee: 2 Comments to the Author(s) This is the first in-depth study of in-vivo vertebral kinematics during suction feeding in fish. Consequently, the manuscript presents very interesting and novel kinematic data. It can also provide the basis for further research into the mechanics and form-function relationships of the vertebral column during suction feeding. The text is clearly written and nicely illustrated in the figures and supplementary videos. Methodologically, I have no concerns as the author is a leading expert in x-ray-based kinematic analyses.
My main criticisms is about the broader framing of the study: the fish vs tetrapods comparison is not clear to me. This concerns the description of the observed motions to be neck-like or the fish's vertebral column to have a functional neck as in tetrapods. As I understood, any vertebra performing some degree of rotation in the sagittal plane during the act of suction feeding are interpreted to be perform a neck-like function. What are the arguments for this view? I'm missing this information, as the function of a neck during feeding in tetrapods has not been defined properly in the text. In terrestrial lower vertebrates like lizards, the neck allows the head and jaws to be aimed and moved towards the food in 3D involving both pitch, yaw, and roll, while the trunk remains stationary (e.g. studies by S. Montuelle). If this would be the reference for neck-like function in a tetrapod, I see more differences than similarities with the current results. Namely, the vertebral column in these fish moves in response to a short blast of high-power muscle activity during the strike at the food, while the lizards manage to stably hold the neck into these 3D postures when approaching the food. So at least the temporal and dynamic aspects of these motions do not seem 'neck-like' with respect to lizards. I fully agree that the vertebral column has an important role for head movement during suction feeding. Yet, the author concludes that the vertebral column translates and positions the head, but did not consider the vertebral column to move in response to the dynamics of the head. Especially for the interpretation of the data of Antennarius, this is important. This animal is experiencing a strong external force in the form of suction propulsion. This forward pulling of the head may cause the straightening of the curves spine. Additionally, the clockwise torque on the head will inevitably push down on the first vertebra, an effect that has also been noted in seahorses (ref 29 in the manuscript; and note examples like the fused four cranialmost vertebra of the powerful head rotator Fistularia seems a response to avoid a collapse of this vertebral region). With this in mind, I'm unsure whether claiming that the data show that the vertebra are translating and positioning the head is appropriate. In other words, I'm afraid that readers will interpret this as an active process driven entirely from within the vertebral musculoskeletal system (statements such as line 59: 'vertebral column producing neck-like motions'), as during slow neck movement in humans, while this is not the case.
(2) Line 27: final sentence -has been written quite a few times now for the muscles surrounding these vertebra and the pectoral girdle. Are you sure you want to repeat this once more, claiming a 'recast' of our scientific view to this dual function?
(3) Line 37: 'pectoral girdle attaches directly to the cranium'. There may be some exceptions, like eels. How was it for the shark in your previous publication in Proc. R. Soc B? (4) Line 126 and other places in the manuscript: 'dorsoventral rotations'. As 'dorsovertral' is a lineal axis, this may be less appropriate than 'dorsoventral plane rotations' or 'sagittal plane rotations'. It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Do you have any ethical concerns with this paper? No
Comments to the Author I appreciate the author's willingness to consider a reframing of the issues brought by the reviewers and editor. I have no other major comments to make or concerns about the way the comments were addressed. The only additional comment I have is that I think there is a missing word in Line 359. I think it should read "joints in extant and fossil fishes".

Review form: Reviewer 2
Recommendation Accept with minor revision (please list in comments)

Scientific importance: Is the manuscript an original and important contribution to its field? Good
General interest: Is the paper of sufficient general interest? Good Quality of the paper: Is the overall quality of the paper suitable? Excellent Is the length of the paper justified? Yes

Do you have any concerns about statistical analyses in this paper? If so, please specify them explicitly in your report. No
It is a condition of publication that authors make their supporting data, code and materials available -either as supplementary material or hosted in an external repository. Please rate, if applicable, the supporting data on the following criteria.

Do you have any ethical concerns with this paper? No
Decision letter (RSPB-2021-1091.R1)

02-Aug-2021
Dear Dr Camp I am pleased to inform you that your manuscript RSPB-2021-1091.R1 entitled "A neck-like vertebral motion in fish" has been accepted for publication in Proceedings B.
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Once again, thank you for submitting your manuscript to Proceedings B and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Decision letter (RSPB-2021-1091.R2)

04-Aug-2021
Dear Dr Camp I am pleased to inform you that your manuscript entitled "A neck-like vertebral motion in fish" has been accepted for publication in Proceedings B.
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Response to Reviewers
Thank you for these helpful and thoughtful responses, and especially the discussion of different mechanisms for producing cranial elevation. I have addressed all the reviewers' and editors' comments and concerns (see below) and in almost all cases implemented their suggested changes in my manuscript (see track-changes document). Below I have copied the original comments (in italics) with my responses and descriptions of the corresponding revisions (in bold). This is followed by a copy of the manuscript with revisions marked as 'tracked changes'.
Associate Editor 1. Both reviewers give the manuscript high marks in general and agree that the data are very valuable, but they raise concerns about the framing and context provided. Most critically, Reviewer 2 questions the functional comparisons between fish and tetrapod necks. What is meant by "neck-like function" should be better defined. In addition, both reviewers request a better explanation of how the head movements are being produced and controlled: by muscular activity? By external forces? Expanding the discussion to address the musculoskeletal anatomy and biomechanical environment of these fish in detail would help to provide a basis for future research in this area. (185-187). These results provide a starting point for investigating how other non-tetrapod vertebrates may use this neck-like, dorsoventral rotation of the vertebral column to move the head relative to the body (lines 233-243).

I am delighted the editor and reviewers found these data valuable. I have revised the framing and context of the study in response to the reviewers' concerns: I now explicitly define the neck-like function this study is considering as dorsoventral flexion of the craniovertebral/intervertebral joints to rotate the head (lines 44-45). And I focus my comparison between fish and tetrapods specifically on the proportion of the vertebral column used to achieve this function (lines 55-61). While tetrapods rely on a cranial region of specialized vertebrae (the neck), trout and frogfish had dorsoventral rotations across large regions of the vertebral column-far beyond the morphologically distinct cervical vertebrae
I agree that how these vertebral motions are produced and controlled is an important topic and added a discussion of possible mechanisms (lines 204-220). These include active shortening of the dorsal body (epaxial) muscles to rotate the head, rotate the vertebrae, or (more likely) both, and the external fluid forces generated by suction feeding (see response #10-11 for more details). And I have provided a brief summary of the notoriously complex epaxial muscle anatomy (lines 207-213) and how it may relate to these vertebral motions. Conclusively determining which mechanisms are used by trout and frogfish and how the complex architecture of the segmented epaxial muscles produce these motions is beyond the scope of this study. This would require substantial new data such epaxial length changes during cranial elevation, and detailed epaxial anatomy--which to my knowledge has never been described in frogfish. However, I have (hopefully!) provided enough information to highlight specific areas of this fascinating system where future research is needed (lines 212-213) and provide a foundation for that work.
Referee: 1 Comments to the Author(s) 2. I commend the author on starting what I hope will be more study of the functional anatomy Appendix A of the anterior vertebrae in fishes. This region of the vertebral column is highly variable across fishes and thus likely plays different roles depending on the group. This paper uses a sophisticated technique to analyze the movement of the anterior vertebrae during feeding in 2 quite distinct fish demonstrating that there are d-v intervertebral movements during feeding. 2. Thank you, I also hope to continue studying the functional morphology of these anterior vertebrae in fishes! 3. Lines 37-38: While the majority of actinopterygian fish have a connection between the pectoral girdle and the school, this is not true for all actinopterygian fishes or for any cartilaginous fishes. It would be good to include some nuance here given this anatomical diversity. 3. I agree completely, and it was certainly not my intention to overlook the remarkable diversity of pectoral girdle anatomy across bony and cartilaginous fishes. I have changed the text here to specify that I am referring to ray-finned fishes and that the pectoral girdle articulates with the skull in most of these fish (lines 37-40). I also now mention the very different pectoral girdle anatomy of sharks in discussing how my results may inform future studies of other groups of fish (lines 236-238).

Lines 93-94:
Were only successful strikes included? Also, how often did strikes include cranial elevation less than 5 degrees? Is there a cranial elevation that did not coincide with vertebral movement? I am trying to get an understanding of how often the vertebral column plays in feeding in these two animals. 4. I have provided more detail on the strikes chosen for analysis in the methods (lines 95-97). All the frogfish and 18 of the 20 trout strikes analysed were successful, which probably reflects the relatively unchallenging prey items: mealworms, pellets, and dead fish or shrimp. I did include two unsuccessful misses from one trout: these were kinematically similar to the successful strikes with relatively large (for these trout) cranial elevations of 10-12 degrees. All frogfish strikes and most (20 out of 30) trout strikes had at least 5 degrees of cranial elevation. I never observed cranial elevation resulting from just rotation of the craniovertebral joint: it was always accompanied by dorsoventral motion of multiple intervertebral joints.
My personal observation is that trout relied less on dorsoventral vertebral rotation and cranial elevation to capture food-this is reflected in the highly variable magnitudes and patterns of these motions (Figs. 2-3 and Supplemental Fig. 3). These fish are excellent swimmers and use both rapid mouth expansion (including cranial elevation) to suck food into the mouth, as well as forward swimming to propel the mouth towards the food (ram feeding). And they adjusted the position and orientation of their whole body in the tank, relative to the food.
In contrast, I suspect vertebral motion is essential for feeding in frogfish. Cranial and vertebral kinematics were surprisingly consistent, compared to trout (Figs. 2-4). These sit-and-wait, benthic predators locomote slowly and rely on massive and rapid mouth expansion to exert suction forces and protrude the jaws towards the food. Dorsoventral vertebral rotation to elevate the head not only directly expands the mouth cavity dorsally, but can contribute to ventral and lateral expansion and jaw protrusion through musculoskeletal linkages in the fish head. And in frogfish the direction of vertebral motion seems linked to the position of the head and mouth relative to the food (Supplemental Fig. 2), rather than moving and re-orienting the whole body like trout.
Of course, these observations come with the caveat that they are based on 3 individuals, in an artificial lab setting, feeding on non-elusive food items.

The author sets up how fish don't have an anatomical neck due to the connection between
the pectoral girdle and skull. However, much of the vertebral movement appears to be posterior to this location. While the author does allude to the functional neck movements being a significant part of the anterior vertebral column, this isn't brought into context with the placement of the pectoral girdle. Would you expect more intervertebral movement in species with no connection of the pectoral girdle to the skull? 5. I agree with the reviewer that the role of pectoral girdle position in neck-like vertebral motions was unclear. It was not my intention to imply that a connection between the skull and pectoral gridle limits intervertebral motion in fish. I now describe the differences in pectoral girdle placement between ray-finned fish and tetrapods in more detail (lines 38-40). Motion between the head and pectoral girdle in ray-finned fish is determined by the joints linking these structures. This is quite different from tetrapods, where the vertebrae of the neck link the head and pectoral girdle. And it suggests that in the "neck" of ray-finned fish, cranio-pectoral mobility may be independent from cranio-vertebral mobility. Because of this, I expect intervertebral movement could be relatively independent of how tightly or loosely the pectoral girdle is connected to the skull, so species without this connection may not necessarily have more vertebral mobility. Also, I now state that the dorsoventral rotations of the vertebral column extend far beyond the pectoral girdle (line 181), and have added the pectoral girdle to Fig. 1 to show its position relative to the vertebral column. While investigating the motion of the pectoral girdle relative to the skull and vertebral column is beyond the scope of this study, it is an intriguing question that I hope will be tackled in future work.
6. It would be great for the author to discuss how the movements of the vertebrae are possibly being controlled given that the myomeres responsible for moving these vertebrae span multiple segments. What does the muscular anatomy look like in this region of the body? 6. I agree that Referee: 2 Comments to the Author(s) 7. This is the first in-depth study of in-vivo vertebral kinematics during suction feeding in fish. Consequently, the manuscript presents very interesting and novel kinematic data. It can also provide the basis for further research into the mechanics and form-function relationships of the vertebral column during suction feeding. The text is clearly written and nicely illustrated in the figures and supplementary videos. Methodologically, I have no concerns as the author is a leading expert in x-ray-based kinematic analyses. 7. Thank you! I'm glad you found the data interesting and the writing and images clear and helpful.

8.
My main criticisms is about the broader framing of the study: the fish vs tetrapods comparison is not clear to me. This concerns the description of the observed motions to be neck-like or the fish's vertebral column to have a functional neck as in tetrapods. As I understood, any vertebra performing some degree of rotation in the sagittal plane during the act of suction feeding are interpreted to be perform a neck-like function. What are the arguments for this view? I'm missing this information, as the function of a neck during feeding in tetrapods has not been defined properly in the text. In terrestrial lower vertebrates like lizards, the neck allows the head and jaws to be aimed and moved towards the food in 3D involving both pitch, yaw, and roll, while the trunk remains stationary (e.g. studies by S. Montuelle). If this would be the reference for neck-like function in a tetrapod, I see more differences than similarities with the current results. Namely, the vertebral column in these fish moves in response to a short blast of high-power muscle activity during the strike at the food, while the lizards manage to stably hold the neck into these 3D postures when approaching the food. So at least the temporal and dynamic aspects of these motions do not seem 'neck-like' with respect to lizards. 8. I agree that the original manuscript did not make it clear exactly what functions and motions of the neck were being compared between fish and tetrapods. I now specify that this study is focusing on just one function of the neck, namely the ability to rotate the head dorsoventrally in the sagittal plane, relative to the body (lines 47-48). This is consistent with previous definitions of a 'functional neck' and 'neck-bending' in fish Additionally, I focus the comparison between fish and tetrapods on what portion of the vertebral column is used to produce dorsoventral head rotation (lines 63-71). This includes highlighting how trout and frogfish differ from tetrapods: rather than using solely the cranial-most, "cervical" vertebrae-analogous to the tetrapod neck-they rotate a much larger region of the vertebral column during cranial elevation (line 185-187). This distinction supports the idea that fish use large regions of the vertebral column for both feeding and swimming, as opposed to using just cranial vertebrae for dorsoventral rotation in feeding and just caudal vertebrae for lateral rotation in swimming. And it suggests that future studies investigating "functional necks" in nontetrapods should consider more caudal vertebrae and not just the craniovertebral joint (lines 318-319). 10. I agree that these data show how the neurocranium and vertebral column are moving, but cannot determine the mechanisms driving these motions. I have revised the text throughout the manuscript to avoid implying that cranial motion is entirely produced by activation of the vertebral musculoskeletal system. And in discussing how these vertebral motions could be generated, I explicitly state that the epaxials could be acting on the head to rotate the vertebrate or vice versa or (what I suspect is most likely) some combination of both (lines 205-207). I have cited the seahorse system as an example (Van Wassenbergh et al., 2011), but was unable to find references for the cranial rotation and morphology of Fistularia. If the reviewer can suggest any literature on this species that I may have missed, I would be grateful and happy to reference them.

Lesiuk
Minor remarks: 11. Line 26: word 'of' should be deleted. 11. Thank you for spotting this error, I have fixed it.

12.
Line 27: final sentencehas been written quite a few times now for the muscles surrounding these vertebra and the pectoral girdle. Are you sure you want to repeat this once more, claiming a 'recast' of our scientific view to this dual function?