Cranial ontogeny of Thamnophis radix (Serpentes: Colubroidea) with a re-evaluation of current paradigms of snake skull evolution

Accurate knowledge of skeletal ontogeny in extant organisms is crucial in understanding important morpho-functional systems and in enabling inferences of the ontogenetic stage of fossil specimens. However, detailed knowledge of skeletal ontogeny is lacking for most squamates, including snakes. Very few studies have discussed postnatal development in snakes, with none incorporating data from all three major ontogenetic stages—embryonic, juvenile and adult. Here, we provide the first analysis encompassing these three ontogenetic stages for any squamate, using the first complete micro-computed tomography (micro-CT)-based segmentations of any non-adult snake, based on fresh specimens of Thamnophis radix. The most significant ontogenetic changes involve the feeding apparatus, with major elongation of the tooth-bearing elements and jaw suspensorium causing a posterior shift in the jaw articulation. This shift enables macrostomy (large-gaped feeding in snakes) and occurs in T. radix via a different developmental trajectory than in most other macrostomatans, indicating that the evolution of macrostomy is more complex than previously thought. The braincase of T. radix is also evolutionarily unique among derived snakes in lacking a crista circumfenestralis, a phenomenon considered herein to represent paedomorphic retention of the embryonic condition. We thus present numerous important challenges to current paradigms regarding snake cranial evolution.


Recommendation?
Major revision is needed (please make suggestions in comments) Comments to the Author(s) Strong et al. present a nice overview of the ontogenetic development of the skull in a modern colubrid snake, Thamnophis sirtalis. I found it intriguing that despite the fact that this is a very well known snake species so much can still be found when closely observing its bones and their development. Things like the contact between quadrate and prootic in the snake embryo and the lack of a crista circumfenestralis in the adult are certainly novel reports and are worth publishing. Having said this, I also found several issues with this manuscript, which preclude publication in its current state.
Here is a summary of the major points: 1) Some of the terminology used is inadequate, like use of terms such as "orbitosphenoidal plate" or "incisive process".
2) There is confusion between fusion of elements and simple sutures.
3) The manuscript would be greatly improved if the lower jaws were rendered separately in lateral and medial view. This way the authors could properly illustrate all the elements they describe, including angular and splenial, and would not have the lower jaws obscuring most of the anatomy of the skull in ventral view (e.g. palatines, parabasisphenoid, and basioccipital cannot be adequately seen because of this). Moreover, the authors took the time to segment all the individual skull bones from the three developmental stages, but then simply figure the whole skulls without showing the internal details of any of the elements. If the authors only wanted to provide colorized versions of the skulls, then Adobe Photoshop could have provided the very same results in a fraction of the time. I suggest that the authors take advantage of the fact that they have digitally segmented individual elements in 3D and provide rendered images to illustrate internal anatomical details that at present are discussed but not figured anywhere (at least as supplementary figures). 4) I noticed an issue with tooth counts. The authors appear to have counted only those teeth that are actually present and ankylosed to the dentigerous elements, when instead they should have provided counts of tooth positions. This resulted in very inconsistent tooth counts (i.e. very low numbers in the juvenile compared to the adult), when in fact the numbers of tooth positions are expected to be very close, if not identical (at least for dentaries and maxillae). Moreover, tooth counts could be affected by intraspecific variability (to a small extent), but the authors provide counts for only 3 of their eight specimens. This way it s not possible to tell whether different tooth counts could be caused by simple random intraspecific variation rather than by ontogenetic change.
5) There is confusion revolving around the concept of paedomorphosis and on how it is detected. The authors assume that if a trait is retained from an embryonic or juvenile stage into the adult stage of a given species then the adult of that species is paedomorphic. This is incorrect. See details below (e.g. Page 21, line 10). This misconception affects their interpretation of the lack of a CCF in Thamnophis as a plesiomorphic feature, and also their interpretation of the upright; however, when the quadrate is elongated in the adult, then it does not remain upright; in fact, a longer quadrate becomes tilted sideways, so that the condyles are approximately in the same vertical transverse plane, but are not aligned vertically one above the other (i.e. the condyles appear to be one above the other only in lateral view). Obviously, a longer quadrate has to be displaced either laterally or posteriorly, or it will create a gap between upper and lower jaws.
Page 18, line 6: I am not sure why the authors think that is the "typical" caenophidian mode. There has been no systematic survey of jaw attachment modes in snakes as far as I am aware, and the references listed here only examined a limited number of species.
Page 19, line 3: "the suspension of the lower jaw shifts from a prootic-supratemporal articulation of the quadrate to a supratemporal-only articulation ( Fig. 3 and 6)." If accurate, this is a very interesting observation. A contact between quadrate and otic capsule is thought to be absent in snake embryos (see for example Kamal and Hammouda (1965:291), who studied the colubroid Psammophis: "It is of importance to notice that the quadrate during ontogeny is quite apart from the neurocranium and bears no connection with the auditory capsule. This is correlated with the absence of the otic process and the subsequent loss of the crista parotica commonly present in Lacertilia. Only in the fully formed chondrocranium, after the quadrate has reached its definitive position, its dorso-medial margin is attached to the hind end of the auditory capsule by the supratemporal (tabular) bone." Kamal, A. M., & Hammouda, H. G. (1965). The development of the skull of Psammophis sibilans. II. The fully formed chondrocranium. Journal of Morphology, 116(2), 247-295.
Because of this, some readers may question the authors' observation. The fact that the quadrate overlaps the prootic in lateral view doesn't mean that they are in contact, let alone in articulation. Furthermore, in Fig. 6 the tilted perspective seems to suggest that the quadrate is only in articulation with the supratemporal in all three developmental stages, and has no contact with the prootic (but could be an artifact of perspective). And finally, if an articulation between quadrate and prootic was present, then one may expect an articulatory facet on the prootic in fig. 5, but such facet is not apparent. I would suggest the authors to provide as supplementary data 3D surface files (in ply or stl format) of all three embryos that have been CT-scanned, in order to unequivocally show that an articulation between quadrate and prootic is indeed consistently present.
Page 19, lines 6-17: Here the authors speculate about the fact that the quadrate in earlier stages of embryonic Thamnophis is likely in articulation with the braincase. As I mentioned above, this goes against what observed by Kamal and Hammouda (1956), and should therefore be discussed in more detail, pointing out the contradiction with that study.
Page 19, line 29: remove "though there are exceptions" (redundant, you already said "in almost all").
Page 19, line 33: "The data present in the T. radix embryos and juveniles reveal that the suspensorium of scolecophidians can be interpreted as heterchronic." I recommend inserting the sentence: ", if representative of the plesiomorphic embryonic snake condition," just after "juveniles". Paedomorphosis is inferred after observing plesiomorphic juvenile features in adults, thus adult scolecophidians can be considered paedomorphs only if the plesiomorphic embryonic condition for snakes matches what described in Thamnophis rather than what described in other snakes (such as Psammophis, the snake described by Kamal and Hammouda [1965]).
Page 19, line 47: replace "juvenile" with "early embryonic". Such anterior orientation in snakes is only visible in developmental stages occurring before those investigated here by the authors; see Kamal & Hammouda (1965) and Boughner et al. (2007).
Page 21, line 10: remove "thus" ("thus" implies that the following sentence is the only possible consequence of what is stated before, but in fact the authors later present another scenario where T. radix is still within colubroidea but is not necessarily the only colubroid without a CCF). Page 21, line 10 and following: "Given that the CCF is absent throughout the embryonic and juvenile forms (Fig. 5), the continued absence of this feature in the adult snake is the result of paedomorphosis, i.e., retention of an embryonic or juvenile feature in an adult individual." This is a misinterpretation of the concept of paedomorphosis. Paedomorphosis is not inferred by the retention of a feature from juvenile to adult, but by the retention in an adult of what is generally assumed to be a juvenile ancestral feature (i.e. the plesiomorphic juvenile condition). In order to say that the absence of a CCF is a juvenile feature, the authors should first show that (at least closely related) juvenile snakes typically lack it (not just Thamnophis). This would identify the plesiomorphic condition (ideally with support from a phylogeny). Then, and only then, they could argue that the absence of a CCF in adult Thamnophis is a paedomorphic feature. Otherwise anything could be a paedomorphic feature, even the presence of two eyes. To quote from McNamara (1986:5): "If a descendant passes through fewer stages of ontogenetic development than its ancestor, the descendant adult form will have morphological characteristics which occurred in juveniles of the ancestor. This phenomenon has been termed paedomorphosis." Or using Klingenberg's (1998:83) words: "The morphological outcomes of changes in rates and timing of development are paedomorphosis or peramorphosis; they are identified by comparisons of ancestors and descendants in relation to the ancestral ontogeny. A descendant is paedomorphic if its later ontogenetic stages retain characteristics from earlier stages of an ancestor".
Page 22, line 19: "A well-studied example of absence of the CCF occurs in Acrochordus, the sister group to the Colubroidea (Fig. 7)." This is the first reference to figure 7, where a summary of the distribution of the CCF and type of macrostomy is presented. However, the authors do not tell anywhere where they have obtained their data. Their material and methods section does not list representatives of all these families and subfamilies of snakes, and to my knowledge there is no published study containing an exhaustive sample of all these taxa and covering their type of jaw suspension. It seems that the authors here are trying to overgeneralize statements presented in the literature, extending observations that were made on some specific booids and colubroids to the whole snake clade, with specifics that go down to each family and subfamily where no data currently exist. The authors referenced a study (their ref. 6) that provided descriptions of the types of macrostomy present in some booids and some colubroids. However, fig. 7 in this manuscript seems to extrapolate the data presented in that work to ALL booids and ALL colubroids, specifying even the condition for every subfamily, but without providing any sources for this additional information. A few examples: 1) How do the authors know that all Dipsadinae show type B macrostomy? Did they personally observe representatives of all species from the subfamily Dipsadinae? If so, why there is no mention of that in their material and methods?
2) The authors themselves seem to recognize the possibility that type C macrostomy may be more widespread than currently known, making the evolution of the macrostomatan condition more complex than previously anticipated (page 25, lines [6][7][8][9][10][11][12], and yet they provide a misleading figure where this is not the case, and type C macrostomy is limited to only two exceptions in a deceivingly very informative image (see also the comment on the caption for Fig. 6).
In conclusion, unless the authors really posses data on the type of suspensorium for all snakes, I would recommend them to remove such level of detail from the figure, which is highly misleading about what we actually know about the distribution of types of macrostomy within specific snake lineages. The authors should simplify this figure and make it very clear that it only represents a summary of their personal hypothesis of the distribution of these traits within snakes. They should be more straightforward about what represents actual data and what is only assumed to happen in the various lineages.
Page 22, line 33 and following: "However, this basal placement of Acrochordus based on CCF morphology was later rejected, due largely to the strong phylogenetic evidence placing Acrochordus at a far more derived position as the sister to Colubroidea [33]; again, this rejection parallels our rejection of scenario one in Thamnophis. This later analysis re-characterized the absence of the CCF as a paedomorphic feature, making Acrochordus unique among snakes in retaining this embryonic state -similar to our scenario two -though lacked ontogenetic data supporting this conclusion [33]." To be honest, Rieppel 1980 (ref. 33 herein) did not discuss the CCF of Acrochordus, but the undivided fissura metotica of this taxon (there is no mention of the CCF at all in that paper). In fact, the basal placement of Acrochordus by McDowell (1979) and others was mostly based on its undivided fissura metotica (a well documented embryonic feature within squamata), and only partially on its lack of a CCF (which according to some is still present in this snake but in rudimentary form, see authors' ref. 27).
Page 23, line 26: "From our data, the persistent absence of the CCF throughout all three ontogenetic stages unequivocally demonstrates this condition in Thamnophis to be a case of paedomorphosis." As I pointed out above (comment on page 21, line 10), this is an incorrect statement based on a misinterpretation of what paedomorphosis is and how it is detected. Absence of a feature in all developmental stages of the same given species does not provide evidence of paedomorphosis. The authors need to identify the plesiomorphic embryonic/juvenile snake condition via a comparison with other embryonic/juvenile snakes.
Page 23, line 43: replace "the adult condition matches the embryonic condition" with "the adult condition matches the plesiomorphic snake embryonic condition".
Page 25, line 3: insert "main" after "two" (exceptions have already been noted in the literature, and have also been mentioned in this manuscript).
Page 25, line 8: insert a reference after Homalopsis (this taxon was not the object of this study).
Page 25, line 12: "Our observations of suspensorium development in T. radix also provide novel evidence for paedomorphosis in the evolution and ontogeny of the scolecophidian skull." The condition observed in Thamnophis does not provide any evidence for paedomorphosis in scolecophidians, unless it is shown to represent the general (plesiomorphic) embryonic snake condition. What it does is open the possibility to new interpretations on how the scolecophidian skull may have come to be if the contact between quadrate and prootic indeed turns out to be a plesiomorphic embryonic snake trait (i.e. pending further data).
Page 25, line 27 and following: "Furthermore, the absence of a crista circumfenestralis in T. radix […] provides evidence for paedomorphosis in the evolution of this trait." As discussed above, the absence of a crista circumfenestralis in embryonic and adult Thamnophis does not demonstrate that paedomorphosis is the cause of this absence in the latter. Thamnophis alone does not tell us anything about which evolutionary developmental process is responsible for the absence of the CCF in adult snakes. The authors first need to verify that absence of a CCF is a plesiomorphic condition in embryonic or juvenile snakes, if that is the case, then they can conclude that its absence in adult Thamnophis can be interpreted as paedomorphic.
Page 33, line 6: as discussed above, in booids the quadrate does not always remain upright (see comment on page page 17, line 47) Reference 25 is likely Kamal (1969) (and not 19696). Dentary & Embryo/Juvenile/Adult: To my knowledge, tooth counts on marginal dentigerous elements (dentary and maxilla) are very conservative in snakes within a given species and throughout ontogeny. Please recheck tooth positions, and make sure you don't simply count visible teeth, but tooth positions. Tooth counts from multiple specimens (and from both sides of the skull) would be welcome to make sure that there is no intraspecific variability added to what may vary ontogenetically. Figure 6: I wonder if showing the angle between quadrate and compound in a tilted perspective of the skull is appropriate (the angles appear smaller than they actually are). I think that showing these angles in lateral view would be much more effective. The figure caption contains a sentence that I find misleading: "this pathway of development is unique to T. radix and one other, unrelated genus within the Caenophidia, indicating independent evolution of this pattern". The sentence assumes that only Thamnophis and another caenophidian have a type C macrostomy (see fig. 7), while in fact those are simply the only two snakes where presence of this type of jaw suspension has been noted so far in the literature. There has been no systematic review of jaw suspension in all snakes, therefore making such generalizations is unjustified. The authors should rephrase by writing that "this pathway of development has so far been observed only in …" (do not write that it is unique to two species, we simply do not know that).

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
This is a well-written paper and was a pleasure to read! I rarely review manuscripts that do not require many grammatical and stylistic changes, so, thank you.
Here are my comments: Materials and Methods section: Briefly explain why you only scanned 8 specimens of this widely available species (cost, local availability?). Were the two juveniles from the same mother or were they unrelated? It is not fatal to your conclusions if the juveniles are related, but it would be good to mention.
You scanned the heads of alcohol preserved specimens, not skulls, so please explain briefly that you digitally removed soft tissues and rendered the skulls for analysis.
Results section 3.1 (page 6, line 15): I think that the premaxillary notch is seen better in the ventral view and you should refer to Fig. 4, not Fig. 3.
Results section 3.5. (page 10, lines 29ff): It would be good to add an illustration of the crista circumfenestralis in a different colubrid to allow direct comparison with your specimens. If that's impossible, insert a citation where the reader could find such an illustration.
Discussion section 4.1 (bottom of page 16): Briefly mention diets of juveniles vs adult T. radix. Since you are talking about ontogentic shifts in diet and gape, prey type is important.
Discussion section 4.3 (page 22): Explain why you didn't examine other Thamnophis to see presence or absence of CCF. This is one of the big points of your paper, you should tell the reader why you didn't check other species. Thamnophis are readily available in collections. The editors assigned to your paper ("Cranial ontogeny of Thamnophis radix (Serpentes: Colubroidea) with a re-evaluation of current paradigms of snake skull evolution") have now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Associate Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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Comments to Author:
Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) Strong et al. present a nice overview of the ontogenetic development of the skull in a modern colubrid snake, Thamnophis sirtalis. I found it intriguing that despite the fact that this is a very well known snake species so much can still be found when closely observing its bones and their development. Things like the contact between quadrate and prootic in the snake embryo and the lack of a crista circumfenestralis in the adult are certainly novel reports and are worth publishing. Having said this, I also found several issues with this manuscript, which preclude publication in its current state.
Here is a summary of the major points: 1) Some of the terminology used is inadequate, like use of terms such as "orbitosphenoidal plate" or "incisive process".
2) There is confusion between fusion of elements and simple sutures.
3) The manuscript would be greatly improved if the lower jaws were rendered separately in lateral and medial view. This way the authors could properly illustrate all the elements they describe, including angular and splenial, and would not have the lower jaws obscuring most of the anatomy of the skull in ventral view (e.g. palatines, parabasisphenoid, and basioccipital cannot be adequately seen because of this). Moreover, the authors took the time to segment all the individual skull bones from the three developmental stages, but then simply figure the whole skulls without showing the internal details of any of the elements. If the authors only wanted to provide colorized versions of the skulls, then Adobe Photoshop could have provided the very same results in a fraction of the time. I suggest that the authors take advantage of the fact that they have digitally segmented individual elements in 3D and provide rendered images to illustrate internal anatomical details that at present are discussed but not figured anywhere (at least as supplementary figures). 4) I noticed an issue with tooth counts. The authors appear to have counted only those teeth that are actually present and ankylosed to the dentigerous elements, when instead they should have provided counts of tooth positions. This resulted in very inconsistent tooth counts (i.e. very low numbers in the juvenile compared to the adult), when in fact the numbers of tooth positions are expected to be very close, if not identical (at least for dentaries and maxillae). Moreover, tooth counts could be affected by intraspecific variability (to a small extent), but the authors provide counts for only 3 of their eight specimens. This way it s not possible to tell whether different tooth counts could be caused by simple random intraspecific variation rather than by ontogenetic change.
5) There is confusion revolving around the concept of paedomorphosis and on how it is detected. The authors assume that if a trait is retained from an embryonic or juvenile stage into the adult stage of a given species then the adult of that species is paedomorphic. This is incorrect. See details below (e.g. Page 21, line 10). This misconception affects their interpretation of the lack of a CCF in Thamnophis as a plesiomorphic feature, and also their interpretation of the scolecophidian skull as the result of paedomorphosis. Because the authors cannot demonstrate that the lack of a CCF or the contact between quadrate and prootic are plesiomorphic embryonic/juvenile traits in snakes, then they cannot reach the conclusion that Thamnophis and scolecophidians are paedomorphic for these traits. The authors can only raise the possibility (i.e. formulate a hypothesis that requires further data to be supported). Figure 7 is quite a problematic figure. It deceivingly suggests that the authors have way more information on the distribution of the various modes of jaw suspension than they actually have. I have extensively discussed the issues relating to this figure in one of my comments below.

6)
Below I provide detailed comments on all major and minor issues as they present themselves in the manuscript: Page 1, line 53: than in most other (insert "most).
Page 6, line 6: "increased integration of the premaxilla with the rest of the snout". I would argue that this is not true. The images show that the premaxilla is very loosely connected to the rest of the snout in all three specimens/stages, and the only contact is between premaxilla and anterior tip of the septomaxilla. Page 6, line 6: the authors mistakenly use the term "incisive process" to refer to the premaxillary process of the vomer (the "incisive process" is a process on the ventral side of the premaxilla of some squamates other than snakes). Please replace "incisive process" with "premaxillary process" here and in any following instance (lines 17, 26). Page 6, line 13: "posterior premaxillary notch which will eventually accommodate the septomaxilla and vomer". The notch does not accommodate any part of those bones. Page 6, line 22: "premaxilla's nasal process expands both dorsoventrally and anteroposteriorly". I believe this is incorrect, it expands mediolaterally and anteroposteriorly. It is quite clear that the relative height of the nasal process remains the same if compared to septomaxilla, nasal, and prefrontal.
Page 6, lines 24-31: "Extension of the anterior processes of the septomaxilla and incisive processes of the vomer relative to the rest of these bones creates stronger contact -and thus improved integration -between the premaxilla and the rest of the snout (Fig. 3C)." This is incorrect, see previous comment on line 6. Page 6, lines 38-39: "dorsal laminae distinctly present and fused to the vertical laminae". This is inaccurate. The dorsal laminae are not individual elements separate from the vertical laminae, they are part of the same element (the nasal), which is in the process of being mineralised. It follows that there can be no fusion between parts of the same element.
Page 7, line 22: The frontals are not completely sutured to each other (don't write "fuse").
Page 7, line 24: please remove "at which point a deep sagittal sulcus is present." (that is not a "sulcus", is the suture between the bones).
Page 7, line 33: I would suggest using the term "optic fenestra" (not "foramen", foramina are small round holes, fenestrae are large gaps, not necessarily round).
Page 7, line 36: Please replace the term "orbitosphenoidal plate" with "descending flange of the frontal". The only literature reference I could find where the term "orbitosphenoid plate" was used in the description of a snake skull dates back to 1831 (and also refers to something different). Please replace "orbitosphenoidal plate" with "descending flange of the frontal" also in the following instances (same page lines 40, 52; and page 8, lines 3, 8).
Page 9, line 22: "The palatine tooth row bears 15 teeth in the embryo, 14 teeth in the juvenile, and 16 teeth in the adult". The palatines are hidden by the lower jaws, so it is impossible to verify the tooth counts from the figures provided. In fact, the lower jaws hide a great deal of the ventral anatomy of the skulls; it would be better to have them rendered separately next to the skulls, this way the authors could also illustrate the medial view of the lower jaws, which at present is not shown. Based on what argued below for the tooth counts on dentaries and maxillae (Page 15, lines 31 & 52), I would suggest the authors to double-check these counts as well, in order to make sure that they actually provide counts of tooth positions and not just of preserved teeth. Finally, the numbers presented here, if correct, do not appear to follow an ontogenetic trend (15-14-16). I suspect that there may be some intraspecific variation in there as well. In order to see if this is the case, the authors should provide tooth counts for all specimens that they CT scanned (3 embryos, 2 juveniles, and 3 adults). With only one specimen per ontogenetic stage it is hard to discriminate between what is actually ontogenetic change and what is simply random intraspecific variation. The same holds for the tooth counts in dentaries and maxillae. Moreover, the authors should also provide the counts for left and right counterparts of each bone, in case the numbers are different.
Page 9, lines 31-35: "The braincase undergoes a general dorsoventral flattening throughout ontogeny, from an initially globular appearance in the embryo to a dorsoventrally compressed and anteroposteriorly elongated form in the adult (Fig. 3A-C)." The compression of the braincase in the adult is mostly anterior, the posterior half is as deep in the adult as it is in the juvenile (the length/max-depth ratio of the skull is about 2.1 in both).
Please be more precise in your description.
Page 9, line 42: insert "posterior to" before "lateral aperture" (the jugular foramen is not posteroventral to the lateral aperture, it is posterior to it).
Page 10, line 19: "The jugular foramen also develops internal struts that subdivides it into three openings internally". "Subdivide" (not "subdivides", the word "struts" is plural), and you could mention which nerves go through those foramina, they are well known in the snake literature.
Page 10, line 36: "stapedial footplate and LARST exposed in lateral view ( Fig. 5A-C)." No stapedial footplate is shown in figure 5, you could add a reference to figure 3 to illustrate your point.
Page 10, lines 42-47: "The nuchal crest that runs anteromedial-posterolateral above the fenestra ovalis is minimally developed in the embryo, resulting in a globular process above each fenestra ovalis". Please add a figure reference and label the nuchal crest in it for those people unfamiliar with the term.
Page 10, line 49: I would replace "this process" with "it".
Page 11, line 36: "a narrowing of the large ventral notch of the supraoccipital". Between the otic capsules? If so, please specify, it is unclear which notch the authors are referring to.
Page 11, lines 40-42: "Internally, the supraoccipital undergoes a general elaboration of internal crests and subdivisions." This sounds extremely vague and superficial. What kind of elaboration? Which crests? Which subdivisions? Please be precise in your description or remove this sentence.
Page 11, line 52: "contact with the prootic is stronger than in the juvenile but still not complete." Do the authors mean: still not complete internally? Externally it looks complete in the figures.
Page 12, line 12 and following: What is the point of segmenting all the individual bones if then internal anatomical details like these are not properly figured and labeled? I would recommend adding some additional figures, if not to the main text at least as supplementary information.
Page 12, line 35 and following: "The junction between the parasphenoid process and the body of the basisphenoid also becomes elaborated, with minor lateral projections in the embryo and juvenile becoming enlarged into prominent triangular projections in the adult. These projections contribute to an increase in sutural contact with the anteroventral parietal." All these details are not visible because hidden by the lower jaws (see also comment above on Page 9, line 22).
Page 13, line 10: delete "dorsal and" (there are no muscle crests on the dorsal surface of these bones).
Page 13, line 19: "a slight dorsal longitudinal crest on the juvenile occipital condyle is extended into a prominent crest running from the centre to the posterior margin of the dorsal basioccipital." I don't quite understand what the authors are describing here, and there is no figure to illustrate this feature. A prominent dorsal longitudinal crest running along the dorsal surface of a snake basioccipital would be a very unusual, if not unique, feature. If this feature is real it should be figured.
Page 13, line 52: same as above, there is no "dorsal mandibular condyle", so the word "ventral" is redundant and can be removed.
Page 13, line 49: "The quadrate also undergoes a progressive deepening of its ventral mandibular condyle". What do the authors mean by "progressive deepening"? it's not clear, a dorsoventral expansion is not apparent form the figures, perhaps a mediolateral expansion?
Page 15, line 6 and following: Here the authors again describe features that are not illustrated. This could be easily remedied by rendering the lower jaws separately in lateral and medial views.
Page 15, line 31: "The dentary bears 18 teeth in the embryo, 18 teeth in the juvenile, and 28 teeth in the adult." These tooth counts cannot be correct. I can count at least 25 tooth positions in the juvenile illustrated in fig. 3. The authors should count tooth positions, not just fully erupted teeth that are ankylosed to the margin of the dentary. As far as I know, tooth counts on the marginal toothbearing elements are very conservative in snake species and across ontogeny, I would be really surprised if Thamnophis would show such differences in tooth counts.
Page 15, line 52: "The maxilla bears 16 teeth in the embryo and 18 teeth in the juvenile and adult." Same problem as above. I can count at least 23 tooth positions in the embryo and a similar number in the juvenile. Please check these numbers again.
Page 16, lines 8-15. Angular and splenial are not figured nor labeled anywhere. Again, the manuscript would be much improved if the lower jaws were rendered separately, so that the ventral aspect of the skull was not obscured, and all elements of the lower jaws could be properly illustrated.
Page 16, lines 19-26: aren't these statolithic masses present in the adults as well? I would be surprised if this was not the case.
Page 17, line 8: "pterygoid and dentary tooth rows, which increase from 18 to 28 and from 22 to 26 teeth, respectively". Based on the likely erroneous tooth counts provided by the authors for dentary and maxilla, I would suggest to check these numbers again.
Page 17, line 47: "the quadrate, though also elongated, remains upright." This is inaccurate. In some booids, such as Calabaria, the quadrate is not elongated and remains upright; however, when the quadrate is elongated in the adult, then it does not remain upright; in fact, a longer quadrate becomes tilted sideways, so that the condyles are approximately in the same vertical transverse plane, but are not aligned vertically one above the other (i.e. the condyles appear to be one above the other only in lateral view). Obviously, a longer quadrate has to be displaced either laterally or posteriorly, or it will create a gap between upper and lower jaws.
Page 18, line 6: I am not sure why the authors think that is the "typical" caenophidian mode. There has been no systematic survey of jaw attachment modes in snakes as far as I am aware, and the references listed here only examined a limited number of species.
Page 19, line 3: "the suspension of the lower jaw shifts from a prootic-supratemporal articulation of the quadrate to a supratemporal-only articulation ( Fig. 3 and 6)." If accurate, this is a very interesting observation. A contact between quadrate and otic capsule is thought to be absent in snake embryos (see for example Kamal and Hammouda (1965:291), who studied the colubroid Psammophis: "It is of importance to notice that the quadrate during ontogeny is quite apart from the neurocranium and bears no connection with the auditory capsule. This is correlated with the absence of the otic process and the subsequent loss of the crista parotica commonly present in Lacertilia. Only in the fully formed chondrocranium, after the quadrate has reached its definitive position, its dorso-medial margin is attached to the hind end of the auditory capsule by the supratemporal (tabular) bone." Because of this, some readers may question the authors' observation. The fact that the quadrate overlaps the prootic in lateral view doesn't mean that they are in contact, let alone in articulation. Furthermore, in Fig. 6 the tilted perspective seems to suggest that the quadrate is only in articulation with the supratemporal in all three developmental stages, and has no contact with the prootic (but could be an artifact of perspective). And finally, if an articulation between quadrate and prootic was present, then one may expect an articulatory facet on the prootic in fig.  5, but such facet is not apparent. I would suggest the authors to provide as supplementary data 3D surface files (in ply or stl format) of all three embryos that have been CT-scanned, in order to unequivocally show that an articulation between quadrate and prootic is indeed consistently present.
Page 19, lines 6-17: Here the authors speculate about the fact that the quadrate in earlier stages of embryonic Thamnophis is likely in articulation with the braincase. As I mentioned above, this goes against what observed by Kamal and Hammouda (1956), and should therefore be discussed in more detail, pointing out the contradiction with that study.
Page 19, line 29: remove "though there are exceptions" (redundant, you already said "in almost all").
Page 19, line 33: "The data present in the T. radix embryos and juveniles reveal that the suspensorium of scolecophidians can be interpreted as heterchronic." I recommend inserting the sentence: ", if representative of the plesiomorphic embryonic snake condition," just after "juveniles". Paedomorphosis is inferred after observing plesiomorphic juvenile features in adults, thus adult scolecophidians can be considered paedomorphs only if the plesiomorphic embryonic condition for snakes matches what described in Thamnophis rather than what described in other snakes (such as Psammophis, the snake described by Kamal and Hammouda [1965]).
Page 19, line 47: replace "juvenile" with "early embryonic". Such anterior orientation in snakes is only visible in developmental stages occurring before those investigated here by the authors; see Kamal & Hammouda (1965)  Page 21, line 10: remove "thus" ("thus" implies that the following sentence is the only possible consequence of what is stated before, but in fact the authors later present another scenario where T. radix is still within colubroidea but is not necessarily the only colubroid without a CCF).
Page 21, line 10 and following: "Given that the CCF is absent throughout the embryonic and juvenile forms (Fig. 5), the continued absence of this feature in the adult snake is the result of paedomorphosis, i.e., retention of an embryonic or juvenile feature in an adult individual." This is a misinterpretation of the concept of paedomorphosis. Paedomorphosis is not inferred by the retention of a feature from juvenile to adult, but by the retention in an adult of what is generally assumed to be a juvenile ancestral feature (i.e. the plesiomorphic juvenile condition). In order to say that the absence of a CCF is a juvenile feature, the authors should first show that (at least closely related) juvenile snakes typically lack it (not just Thamnophis). This would identify the plesiomorphic condition (ideally with support from a phylogeny). Then, and only then, they could argue that the absence of a CCF in adult Thamnophis is a paedomorphic feature. Otherwise anything could be a paedomorphic feature, even the presence of two eyes. To quote from McNamara (1986:5): "If a descendant passes through fewer stages of ontogenetic development than its ancestor, the descendant adult form will have morphological characteristics which occurred in juveniles of the ancestor. This phenomenon has been termed paedomorphosis." Or using Klingenberg's (1998:83) words: "The morphological outcomes of changes in rates and timing of development are paedomorphosis or peramorphosis; they are identified by comparisons of ancestors and descendants in relation to the ancestral ontogeny. A descendant is paedomorphic if its later ontogenetic stages retain characteristics from earlier stages of an ancestor".
Page 22, line 19: "A well-studied example of absence of the CCF occurs in Acrochordus, the sister group to the Colubroidea (Fig. 7)." This is the first reference to figure 7, where a summary of the distribution of the CCF and type of macrostomy is presented. However, the authors do not tell anywhere where they have obtained their data. Their material and methods section does not list representatives of all these families and subfamilies of snakes, and to my knowledge there is no published study containing an exhaustive sample of all these taxa and covering their type of jaw suspension. It seems that the authors here are trying to overgeneralize statements presented in the literature, extending observations that were made on some specific booids and colubroids to the whole snake clade, with specifics that go down to each family and subfamily where no data currently exist. The authors referenced a study (their ref. 6) that provided descriptions of the types of macrostomy present in some booids and some colubroids. However, fig. 7 in this manuscript seems to extrapolate the data presented in that work to ALL booids and ALL colubroids, specifying even the condition for every subfamily, but without providing any sources for this additional information. A few examples: 1) How do the authors know that all Dipsadinae show type B macrostomy? Did they personally observe representatives of all species from the subfamily Dipsadinae? If so, why there is no mention of that in their material and methods?
2) The authors themselves seem to recognize the possibility that type C macrostomy may be more widespread than currently known, making the evolution of the macrostomatan condition more complex than previously anticipated (page 25, lines [6][7][8][9][10][11][12], and yet they provide a misleading figure where this is not the case, and type C macrostomy is limited to only two exceptions in a deceivingly very informative image (see also the comment on the caption for Fig. 6).
In conclusion, unless the authors really posses data on the type of suspensorium for all snakes, I would recommend them to remove such level of detail from the figure, which is highly misleading about what we actually know about the distribution of types of macrostomy within specific snake lineages. The authors should simplify this figure and make it very clear that it only represents a summary of their personal hypothesis of the distribution of these traits within snakes. They should be more straightforward about what represents actual data and what is only assumed to happen in the various lineages.
Page 22, line 33 and following: "However, this basal placement of Acrochordus based on CCF morphology was later rejected, due largely to the strong phylogenetic evidence placing Acrochordus at a far more derived position as the sister to Colubroidea [33]; again, this rejection parallels our rejection of scenario one in Thamnophis. This later analysis re-characterized the absence of the CCF as a paedomorphic feature, making Acrochordus unique among snakes in retaining this embryonic state -similar to our scenario two -though lacked ontogenetic data supporting this conclusion [33]." To be honest, Rieppel 1980 (ref. 33 herein) did not discuss the CCF of Acrochordus, but the undivided fissura metotica of this taxon (there is no mention of the CCF at all in that paper). In fact, the basal placement of Acrochordus by McDowell (1979) and others was mostly based on its undivided fissura metotica (a well documented embryonic feature within squamata), and only partially on its lack of a CCF (which according to some is still present in this snake but in rudimentary form, see authors' ref. 27).
Page 23, line 26: "From our data, the persistent absence of the CCF throughout all three ontogenetic stages unequivocally demonstrates this condition in Thamnophis to be a case of paedomorphosis." As I pointed out above (comment on page 21, line 10), this is an incorrect statement based on a misinterpretation of what paedomorphosis is and how it is detected. Absence of a feature in all developmental stages of the same given species does not provide evidence of paedomorphosis. The authors need to identify the plesiomorphic embryonic/juvenile snake condition via a comparison with other embryonic/juvenile snakes.
Page 23, line 43: replace "the adult condition matches the embryonic condition" with "the adult condition matches the plesiomorphic snake embryonic condition".
Page 25, line 3: insert "main" after "two" (exceptions have already been noted in the literature, and have also been mentioned in this manuscript).
Page 25, line 8: insert a reference after Homalopsis (this taxon was not the object of this study).
Page 25, line 12: "Our observations of suspensorium development in T. radix also provide novel evidence for paedomorphosis in the evolution and ontogeny of the scolecophidian skull." The condition observed in Thamnophis does not provide any evidence for paedomorphosis in scolecophidians, unless it is shown to represent the general (plesiomorphic) embryonic snake condition. What it does is open the possibility to new interpretations on how the scolecophidian skull may have come to be if the contact between quadrate and prootic indeed turns out to be a plesiomorphic embryonic snake trait (i.e. pending further data).
Page 25, line 27 and following: "Furthermore, the absence of a crista circumfenestralis in T. radix […] provides evidence for paedomorphosis in the evolution of this trait." As discussed above, the absence of a crista circumfenestralis in embryonic and adult Thamnophis does not demonstrate that paedomorphosis is the cause of this absence in the latter. Thamnophis alone does not tell us anything about which evolutionary developmental process is responsible for the absence of the CCF in adult snakes. The authors first need to verify that absence of a CCF is a plesiomorphic condition in embryonic or juvenile snakes, if that is the case, then they can conclude that its absence in adult Thamnophis can be interpreted as paedomorphic.
Page 33, line 6: as discussed above, in booids the quadrate does not always remain upright (see comment on page page 17, line 47) Reference 25 is likely Kamal (1969) (and not 19696). Table 1: Frontal & Adult: Completely sutured with frontal (not "completely fused along frontal suture"). Parietal & Embryo: "Almost entirely unfused along sagittal suture." There is no sagittal suture to speak of, only an unossified central region of the parietal. Parietal & Juvenile: parietal roof ossified (not "sutured").
Dentary & Embryo/Juvenile/Adult: To my knowledge, tooth counts on marginal dentigerous elements (dentary and maxilla) are very conservative in snakes within a given species and throughout ontogeny. Please recheck tooth positions, and make sure you don't simply count visible teeth, but tooth positions. Tooth counts from multiple specimens (and from both sides of the skull) would be welcome to make sure that there is no intraspecific variability added to what may vary ontogenetically. Figure 6: I wonder if showing the angle between quadrate and compound in a tilted perspective of the skull is appropriate (the angles appear smaller than they actually are). I think that showing these angles in lateral view would be much more effective. The figure caption contains a sentence that I find misleading: "this pathway of development is unique to T. radix and one other, unrelated genus within the Caenophidia, indicating independent evolution of this pattern". The sentence assumes that only Thamnophis and another caenophidian have a type C macrostomy (see fig. 7), while in fact those are simply the only two snakes where presence of this type of jaw suspension has been noted so far in the literature. There has been no systematic review of jaw suspension in all snakes, therefore making such generalizations is unjustified. The authors should rephrase by writing that "this pathway of development has so far been observed only in …" (do not write that it is unique to two species, we simply do not know that).

Reviewer: 2
Comments to the Author(s) This is a well-written paper and was a pleasure to read! I rarely review manuscripts that do not require many grammatical and stylistic changes, so, thank you.
Here are my comments: Materials and Methods section: Briefly explain why you only scanned 8 specimens of this widely available species (cost, local availability?). Were the two juveniles from the same mother or were they unrelated? It is not fatal to your conclusions if the juveniles are related, but it would be good to mention.
You scanned the heads of alcohol preserved specimens, not skulls, so please explain briefly that you digitally removed soft tissues and rendered the skulls for analysis.

Recommendation? Accept as is
Comments to the Author(s) I must confess that I am a bit disappointed by the tone of the authors' replies, and especially that after spending a considerable amount of time writing what I would consider a thorough and helpful review, my comments were considered "unrealistic, demanding, and often pedantic". I can guarantee the authors that it was not my intention "to create the impression that their manuscript was flawed empirically, conceptually, and grammatically." A review is meant to provide comments and suggestions on how to improve a manuscript, and my belief is that it needs to be rigorous when pointing out flaws, no matter how small.
In any case, the manuscript has now been significantly improved, and I have only a few additional comments. 1) My original comment on Page 6, line 13 was: "posterior premaxillary notch which will eventually accommodate the septomaxilla and vomer". The notch does not accommodate any part of those bones. (2008:pg 404, fig. 2.25). Perhaps we are referring to different notches, but as mentioned, we illustrate what we mean by this.

To which the authors replied: The Reviewer is in error -the premaxilla of all squamates, Thamnophis included, articulates with both the vomer and septomaxilla. The articulation is clearly articulated and the notch we are discussing is visible in our supplementary files (S4-S6), and in numerous illustrations provided in such works as Cundall and Irish
I take it that the posterior notch in question is the notch between the two vomerine processes of the premaxilla, if so, it does not accommodate neither the vomer nor the septomaxilla. While the vomer and septomaxilla do articulate with the premaxilla in most squamates, these bones do not enter that notch (quite obvious in Fig. 4). If the authors are referring to a different notch then please elaborate and be more precise in your description.

2) My original comment on Page 12, line 12 was: What is the point of segmenting all the individual bones if then internal anatomical details like these are not properly figured and labeled? I would recommend adding some additional figures, if not to the main text at least as supplementary information.
To which the authors replied: We have added a figure showing an internal view of the braincase, with pertinent features labelled (Fig. 6D-F). Had the Reviewer consulted the supplementary data he/she would have noted we supplied the requested internal anatomy as STL files.
I must admit that I missed the supplementary data in my first round of review, but in my defense there was no mention of such data in the main text (the link to the dryad repository was only presented at the end of the manuscript and could be easily missed). In any case, anatomical features are not labeled in the STL files, therefore the new figures certainly help illustrate what the authors are writing about.

3) Concerning Fig. 7, my original comment was: In conclusion, unless the authors really posses data on the type of suspensorium for all snakes, I would recommend them to remove such level of detail from the figure, which is highly misleading about what we actually know about the distribution of types of macrostomy within specific snake lineages. The authors should simplify this figure and make it very clear that it only represents a summary of their personal hypothesis of the distribution of these traits within snakes. They should be more straightforward about what represents actual data and what is only assumed to happen in the various lineages.
To which the authors replied: Strongly disagreed. The reviewer cannot be serious on this comment. Again, establishing the nonsense empirical constraint of having ALL data on ALL snakes does not make this requirement so, it only enforces the Reviewer's misguided approach to this component of our manuscript. A case in point, no phylogenetic hypothesis ever published, using genes or morphology, has ever once sampled all taxa and all genes and all morphology -never once; according to the Reviewer's expectations of our study, not a single phylogenetic or comparative study should ever have been published in the history of evolutionary biology. Yet these hypotheses of sister-group relationships and inferred phylogeny exist and are used, clearly by the Reviewer (if in fact the reviewer is not guilty of generating phylogenies from incomplete and imperfect data sampling) for second, third and fourth order hypothesis constructions. It is ridiculous to make such statements and expect accommodation of these extensive, misguided, and condescending criticisms.
Obviously I would not expect anyone to sample all species of snakes to draw some general conclusions as those presented in this manuscript, mine was a rhetorical point. The issue was that the authors' Fig. 7 did not make it explicit which taxa had been actually sampled and for which ones anatomical conditions had been extrapolated. A naïve reader (or just someone unfamiliar with the snake literature) looking at their figure may have mistakenly concluded that all of those snake families and subfamilies had been sampled, at least partially if not thoroughly. I am pleased to see that the authors have modified their Fig. 7 so that readers can now see which families/subfamilies have actually been sampled, and also made it explicit that the figure just represents their personal hypothesis of the evolution of macrostomy.

4) My original comment on Page 22, line 33 was: To be honest, Rieppel 1980 (ref. 33 herein) did not discuss the CCF of Acrochordus, but the undivided fissura metotica of this taxon (there is no mention of the CCF at all in that paper).
To which the authors replied: Note that, while Rieppel (1980) focussed on the undivided metotic fissure as paedomorphic, his later paper with Zaher [ref. 18 herein] included the CCF in this hypothesis of paedomorphosis: "[Acrochordus] appears to retain a number of surprisingly plesiomorphic features in its cranial anatomy. [...] The most unusual features in the skull of Acrochordus are the apparent lack of the crista circumfenestralis and the undivided metotic fissure (McDowell, 1979), characteristics that have been explained as a probably consequence of paedomorphosis." (Rieppel and Zaher 2001: pg 252). As such, our discussion of Rieppel (1980) similarly mentions the overall otico-occipital region of Acrochordus as paedomorphic, in line with Rieppel and Zaher's (2001)

discussion of this hypothesis.
Then the authors should have referenced Rieppel and Zaher (2001) and not Rieppel (1980). However, the authors should know that Rieppel and Zaher (2001) also misquoted Rieppel (1980). In fact, the complete quote from Rieppel and Zaher (2001:252) would be "In light of these phylogenetic relationships, the most unusual features in the skull of Acrochordus are the apparent lack of the crista circumfenestralis and the undivided metotic fissure (McDowell, 1979), characteristics that have been explained as a probable consequence of paedomorphosis (Rieppel, 1980)." The citation of Rieppel (1980) at the end of the quote was conveniently left out by the authors in their reply. I suspect that this is a case where a misquote is reiterated by someone (the authors) who simply trusted another paper (in this case Rieppel and Zaher, 2001) without actually verifying the accuracy of that quote by reading the original source (in this case Rieppel, 1980). Thus, a situation is created where two articles (Rieppel and Zaher, 2001 and this manuscript) make reference to the work of Rieppel (1980) as if it said something about the CCF of Acrochordus as being a paedomorphic feature, while in truth that work didn't mention the CCF at all. I understand that it is common practice to trust what is written in the primary literature and take the references therein as accurate (especially when one of the authors is on both the referenced and the citing paper!). However, the authors should have checked the original paper by Rieppel to make sure that their statements are indeed accurate and avoid perpetuating a case of poor scholarship.
Despite all this, I am happy with the way the authors have modified the problematic paragraph so that a reference to Rieppel (1980) implying that he wrote about the CCF of Acrochordus is now avoided: We have modified this paragraph as follows: "The absence of the CCF in Acrochordus was therefore considered consistent with a basal placement of Acrochordus among snakes, similar to the first scenario proposed herein for Thamnophis [36]. However, this basal placement of Acrochordus was later rejected, due largely to the strong phylogenetic evidence placing Acrochordus at a far more deeply nested position as the sister to Colubroidea [37]; again, this rejection parallels our rejection of scenario one in Thamnophis (i.e., lack of a CCF does not inherently indicate basal phylogenetic status). This later analysis re-characterized the otico-occipital region of the skull of Acrochordus -focussing on he persistent lack of division of the metotic fissure -as paedomorphic, making Acrochordus unique among snakes in retaining this embryonic morphology -similar to our scenario two -though lacked ontogenetic data supporting this conclusion [18,37]." In summary, I would recommend the authors to be more explicit about which "posterior premaxillary notch" (point 1 above) they are referring to, because at the moment the only posterior premaxillary notch I can think of is that between the vomerine processes of the premaxilla. This notch does not accommodate the septomaxilla or the vomer (quite evident in their Fig 4). If the notch they are describing is another notch, then they need to be clearer in their description. Apart from this minor issue I am happy with the manuscript as it is.

04-Jul-2019
Dear Ms Strong: On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-182228.R1 entitled "Cranial ontogeny of Thamnophis radix (Serpentes: Colubroidea) with a re-evaluation of current paradigms of snake skull evolution" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
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Kind regards, Alice Power Editorial Coordinator Royal Society Open Science openscience@royalsociety.org on behalf of Kevin Padian (Subject Editor) openscience@royalsociety.org Associate Editor Comments to Author: A few remaining comments have been made by the reviewer (who is broadly happy with the paper now), which we would like you to take into consideration and modify the manuscript accordingly. We'll look forward to receiving the revision shortly.
Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) I must confess that I am a bit disappointed by the tone of the authors' replies, and especially that after spending a considerable amount of time writing what I would consider a thorough and helpful review, my comments were considered "unrealistic, demanding, and often pedantic". I can guarantee the authors that it was not my intention "to create the impression that their manuscript was flawed empirically, conceptually, and grammatically." A review is meant to provide comments and suggestions on how to improve a manuscript, and my belief is that it needs to be rigorous when pointing out flaws, no matter how small.
In any case, the manuscript has now been significantly improved, and I have only a few additional comments. 1) My original comment on Page 6, line 13 was: "posterior premaxillary notch which will eventually accommodate the septomaxilla and vomer". The notch does not accommodate any part of those bones.
To which the authors replied: The Reviewer is in error -the premaxilla of all squamates, Thamnophis included, articulates with both the vomer and septomaxilla. The articulation is clearly articulated and the notch we are discussing is visible in our supplementary files (S4-S6), and in numerous illustrations provided in such works as Cundall and Irish (2008:pg 404, fig. 2.25). Perhaps we are referring to different notches, but as mentioned, we illustrate what we mean by this.
I take it that the posterior notch in question is the notch between the two vomerine processes of the premaxilla, if so, it does not accommodate neither the vomer nor the septomaxilla. While the vomer and septomaxilla do articulate with the premaxilla in most squamates, these bones do not enter that notch (quite obvious in Fig. 4). If the authors are referring to a different notch then please elaborate and be more precise in your description.

2) My original comment on Page 12, line 12 was: What is the point of segmenting all the individual bones if then internal anatomical details like these are not properly figured and labeled? I would recommend adding some additional figures, if not to the main text at least as supplementary information.
To which the authors replied: We have added a figure showing an internal view of the braincase, with pertinent features labelled (Fig. 6D-F). Had the Reviewer consulted the supplementary data he/she would have noted we supplied the requested internal anatomy as STL files.
I must admit that I missed the supplementary data in my first round of review, but in my defense there was no mention of such data in the main text (the link to the dryad repository was only presented at the end of the manuscript and could be easily missed). In any case, anatomical features are not labeled in the STL files, therefore the new figures certainly help illustrate what the authors are writing about. To which the authors replied: Strongly disagreed. The reviewer cannot be serious on this comment. Again, establishing the nonsense empirical constraint of having ALL data on ALL snakes does not make this requirement so, it only enforces the Reviewer's misguided approach to this component of our manuscript. A case in point, no phylogenetic hypothesis ever published, using genes or morphology, has ever once sampled all taxa and all genes and all morphology -never once; according to the Reviewer's expectations of our study, not a single phylogenetic or comparative study should ever have been published in the history of evolutionary biology. Yet these hypotheses of sister-group relationships and inferred phylogeny exist and are used, clearly by the Reviewer (if in fact the reviewer is not guilty of generating phylogenies from incomplete and imperfect data sampling) for second, third and fourth order hypothesis constructions. It is ridiculous to make such statements and expect accommodation of these extensive, misguided, and condescending criticisms.
Obviously I would not expect anyone to sample all species of snakes to draw some general conclusions as those presented in this manuscript, mine was a rhetorical point. The issue was that the authors' Fig. 7 did not make it explicit which taxa had been actually sampled and for which ones anatomical conditions had been extrapolated. A naïve reader (or just someone unfamiliar with the snake literature) looking at their figure may have mistakenly concluded that all of those snake families and subfamilies had been sampled, at least partially if not thoroughly. I am pleased to see that the authors have modified their Fig. 7 so that readers can now see which families/subfamilies have actually been sampled, and also made it explicit that the figure just represents their personal hypothesis of the evolution of macrostomy. To which the authors replied: Note that, while Rieppel (1980) focussed on the undivided metotic fissure as paedomorphic, his later paper with Zaher [ref. 18 herein] included the CCF in this hypothesis of paedomorphosis: "[Acrochordus] appears to retain a number of surprisingly plesiomorphic features in its cranial anatomy. [...] The most unusual features in the skull of Acrochordus are the apparent lack of the crista circumfenestralis and the undivided metotic fissure (McDowell, 1979), characteristics that have been explained as a probably consequence of paedomorphosis." (Rieppel and Zaher 2001: pg 252). As such, our discussion of Rieppel (1980) similarly mentions the overall otico-occipital region of Acrochordus as paedomorphic, in line with Rieppel and Zaher's (2001) discussion of this hypothesis.

4) My original comment on
Then the authors should have referenced Rieppel and Zaher (2001) and not Rieppel (1980). However, the authors should know that Rieppel and Zaher (2001) also misquoted Rieppel (1980). In fact, the complete quote from Rieppel and Zaher (2001:252) would be "In light of these phylogenetic relationships, the most unusual features in the skull of Acrochordus are the apparent lack of the crista circumfenestralis and the undivided metotic fissure (McDowell, 1979), characteristics that have been explained as a probable consequence of paedomorphosis (Rieppel, 1980)." The citation of Rieppel (1980) at the end of the quote was conveniently left out by the authors in their reply. I suspect that this is a case where a misquote is reiterated by someone (the authors) who simply trusted another paper (in this case Rieppel and Zaher, 2001) without actually verifying the accuracy of that quote by reading the original source (in this case Rieppel, 1980). Thus, a situation is created where two articles (Rieppel and Zaher, 2001 and this manuscript) make reference to the work of Rieppel (1980) as if it said something about the CCF of Acrochordus as being a paedomorphic feature, while in truth that work didn't mention the CCF at all. I understand that it is common practice to trust what is written in the primary literature and take the references therein as accurate (especially when one of the authors is on both the referenced and the citing paper!). However, the authors should have checked the original paper by Rieppel to make sure that their statements are indeed accurate and avoid perpetuating a case of poor scholarship.
Despite all this, I am happy with the way the authors have modified the problematic paragraph so that a reference to Rieppel (1980) implying that he wrote about the CCF of Acrochordus is now avoided: We have modified this paragraph as follows: "The absence of the CCF in Acrochordus was therefore considered consistent with a basal placement of Acrochordus among snakes, similar to the first scenario proposed herein for Thamnophis [36]. However, this basal placement of Acrochordus was later rejected, due largely to the strong phylogenetic evidence placing Acrochordus at a far more deeply nested position as the sister to Colubroidea [37]; again, this rejection parallels our rejection of scenario one in Thamnophis (i.e., lack of a CCF does not inherently indicate basal phylogenetic status). This later analysis re-characterized the otico-occipital region of the skull of Acrochordus -focussing on he persistent lack of division of the metotic fissure -as paedomorphic, making Acrochordus unique among snakes in retaining this embryonic morphology -similar to our scenario two -though lacked ontogenetic data supporting this conclusion [18,37]." In summary, I would recommend the authors to be more explicit about which "posterior premaxillary notch" (point 1 above) they are referring to, because at the moment the only posterior premaxillary notch I can think of is that between the vomerine processes of the premaxilla. This notch does not accommodate the septomaxilla or the vomer (quite evident in their Fig 4). If the notch they are describing is another notch, then they need to be clearer in their description. Apart from this minor issue I am happy with the manuscript as it is.

17-Jul-2019
Dear Ms Strong, I am pleased to inform you that your manuscript entitled "Cranial ontogeny of Thamnophis radix (Serpentes: Colubroidea) with a re-evaluation of current paradigms of snake skull evolution" is now accepted for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial office (openscience_proofs@royalsociety.org and openscience@royalsociety.org) to let us know if you are likely to be away from e-mail contact. Due to rapid publication and an extremely tight schedule, if comments are not received, your paper may experience a delay in publication.
Royal Society Open Science operates under a continuous publication model (http://bit.ly/cpFAQ). Your article will be published straight into the next open issue and this will be the final version of the paper. As such, it can be cited immediately by other researchers. As the issue version of your paper will be the only version to be published I would advise you to check your proofs thoroughly as changes cannot be made once the paper is published.

To the Editor:
Prior to responding to Reviewer 1, we feel it is important to respectfully convey our concerns regarding aspects of this review. Reviewer 1 did provide many useful comments and criticisms of our manuscript, which we greatly appreciate and which improved the document; however, we also feel that in many places the review is unrealistic and demanding, and often pedantic to the point where it seems the Reviewer wanted merely to create the impression that our manuscript was flawed empirically, conceptually, and grammatically. As is often the case with such reviews, Reviewer 1 is also mistaken on many points of grammar, anatomy, and interpretation. We have accommodated this Reviewer's comments to the best of our ability, though we have also rebutted numerous points where necessary with fully reasoned arguments.
As evidence of this broad problem in Review 1, we point to the nature and tone of the second review to illustrate our point.
Nevertheless, as mentioned previously and in the spirit of the peer review process, we have made corrections and thus improvements to the manuscript, rebutting only those comments/criticisms which we considered incorrect.

Reviewers' Comments to Author:
Reviewer: 1 Comments to the Author(s) Strong et al. present a nice overview of the ontogenetic development of the skull in a modern colubrid snake, Thamnophis sirtalis. I found it intriguing that despite the fact that this is a very well known snake species so much can still be found when closely observing its bones and their development. Things like the contact between quadrate and prootic in the snake embryo and the lack of a crista circumfenestralis in the adult are certainly novel reports and are worth publishing. Having said this, I also found several issues with this manuscript, which preclude publication in its current state.
We must point out immediately that we are not presenting information on Thamnophis sirtalis, but rather Thamnophis radix (the Plains Garter Snake). We compare our materials to a skeletonized specimen of T. sirtalis but this manuscript details new data from T. radix.
Here is a summary of the major points: 1) Some of the terminology used is inadequate, like use of terms such as "orbitosphenoidal plate" or "incisive process".
Agreed. This terminology has been changed, in accordance with the reviewer's suggestions -see specific comments below.
2) There is confusion between fusion of elements and simple sutures.
Agreed. This has also been revised -see specific comments below.

Appendix A
3) The manuscript would be greatly improved if the lower jaws were rendered separately in lateral and medial view. This way the authors could properly illustrate all the elements they describe, including angular and splenial, and would not have the lower jaws obscuring most of the anatomy of the skull in ventral view (e.g. palatines, parabasisphenoid, and basioccipital cannot be adequately seen because of this).
Agreed. We've added a figure (new Fig. 5) showing the skull without the lower jaws in ventral view, with a separate depiction of the lower jaws in dorsal and medial view (lateral and ventral views of the jaws are visible in Fig. 3 and 4, respectively). We decided to add an extra figure instead of simply modifying Fig. 4 so that a ventral view of the complete articulated skull is still included.
Moreover, the authors took the time to segment all the individual skull bones from the three developmental stages, but then simply figure the whole skulls without showing the internal details of any of the elements. If the authors only wanted to provide colorized versions of the skulls, then Adobe Photoshop could have provided the very same results in a fraction of the time. I suggest that the authors take advantage of the fact that they have digitally segmented individual elements in 3D and provide rendered images to illustrate internal anatomical details that at present are discussed but not figured anywhere (at least as supplementary figures).
Disagreed. Surface mesh (.stl) files of each bone from the featured skulls were included in the supplementary data available to the reviewers (https://datadryad.org/review?doi=doi:10.5061/dryad.n50st0n). We also included videos of the skulls that the reader could easily access to obtain this anatomical data. It is clear to us that Reviewer #1 did not refer to our supplementary data files as submitted. However, to ensure that readers are aware of these files, we have added references to the supplementary data in the figure captions. We have also added a figure showing an internal view of the braincase (Fig. 6D-F) in order to illustrate pertinent internal anatomical features. 4) I noticed an issue with tooth counts. The authors appear to have counted only those teeth that are actually present and ankylosed to the dentigerous elements, when instead they should have provided counts of tooth positions. This resulted in very inconsistent tooth counts (i.e. very low numbers in the juvenile compared to the adult), when in fact the numbers of tooth positions are expected to be very close, if not identical (at least for dentaries and msaxillae). Moreover, tooth counts could be affected by intraspecific variability (to a small extent), but the authors provide counts for only 3 of their eight specimens. This way it s not possible to tell whether different tooth counts could be caused by simple random intraspecific variation rather than by ontogenetic change.
Agreed. While we do not consider this to be a major issue with the manuscript as written, we have modified the tooth counts to reflect our best estimate regarding number of tooth positions, along with a count of fully ankylosed teeth.

5)
There is confusion revolving around the concept of paedomorphosis and on how it is detected. The authors assume that if a trait is retained from an embryonic or juvenile stage into the adult stage of a given species then the adult of that species is paedomorphic. This is incorrect. See details below (e.g. Page 21, line 10). This misconception affects their interpretation of the lack of a CCF in Thamnophis as a plesiomorphic feature, and also their interpretation of the scolecophidian skull as the result of paedomorphosis. Because the authors cannot demonstrate that the lack of a CCF or the contact between quadrate and prootic are plesiomorphic embryonic/juvenile traits in snakes, then they cannot reach the conclusion that Thamnophis and scolecophidians are paedomorphic for these traits. The authors can only raise the possibility (i.e. formulate a hypothesis that requires further data to be supported).
We have addressed these points in detail below and modified the manuscript text accordingly -see relevant comments (especially page 19 line 33 and page 21 line 10). It is true that an inference of paedomorphosis in an evolutionary context depends on a comparison with a sister taxon or more. However, we believe nobody challenges the notion that the presence of a fully developed CCF is widespread among colubroids, and that ontogenetically, to get to a fully developed CCF condition an individual must pass through a stage where the CCF is still not fully developed. Even in the absence of an explicit phylogenetic mapping of this particular character among species closely related to Thamnophis, it is safe to assume that the condition in Thamnophis would necessarily be a paedomorphic one compared to almost any other colubroid. We remind the Reviewer we are making a comparison at very broad phylogenetic levels, comparing Thamnophis to the generalized condition among colubroids and other families. This is not a low taxonomic level comparison or analysis (i.e., genus level or species level). However, to try to meet this criticism we have provided the above explanation explicitly in the text and acknowledge that further studies within the genus Thamnophis are necessary to indicate if this pattern is specific to T. radix (therefore a paedomorphic event within the genus), or if it is widespread among other species of the genus (implying that the entire genus is paedomorphic relative to others). Figure 7 is quite a problematic figure. It deceivingly suggests that the authors have way more information on the distribution of the various modes of jaw suspension than they actually have. I have extensively discussed the issues relating to this figure in one of my comments below. Below I provide detailed comments on all major and minor issues as they present themselves in the manuscript:
Done. We have also made another revision to the abstract, modifying page 1 line 44 to specify that these are the first complete micro-CT segmentations of any non-adult snake skull (at the time of initial submission, these were the first complete segmentations of any snake skull, but since then a paper on scolecophidian skull anatomy included a completely segmented adult skull). Page 2, line 17: replace "many" with "some" (there aren't really that many studies).
Disagreed. Making this change would alter the meaning of this sentence. We have modified the wording to: "Of these few studies on snake postnatal cranial ontogeny, many have focused on changes in gape size and diet…". As the reviewer indicates, there haven't been many studies on this topic; however, what we're conveying in this sentence is that, of the few studies that have been done, they mostly focus on gape size and diet. Our addition of the word "few" clarifies this point.
Page 6, line 6: "increased integration of the premaxilla with the rest of the snout". I would argue that this is not true. The images show that the premaxilla is very loosely connected to the rest of the snout in all three specimens/stages, and the only contact is between premaxilla and anterior tip of the septomaxilla.
We have no response to this point other to disagree with the Reviewer. We invite the reviewer to access the online supplementary videos and STL files to better observe the differences between the two ontogenetic stages.
Page 6, line 6: the authors mistakenly use the term "incisive process" to refer to the premaxillary process of the vomer (the "incisive process" is a process on the ventral side of the premaxilla of some squamates other than snakes). Please replace "incisive process" with "premaxillary process" here and in any following instance (lines 17, 26).

Done.
Page 6, line 13: "posterior premaxillary notch which will eventually accommodate the septomaxilla and vomer". The notch does not accommodate any part of those bones.
The Reviewer is in error -the premaxilla of all squamates, Thamnophis included, articulates with both the vomer and septomaxilla. The articulation is clearly articulated and the notch we are discussing is visible in our supplementary files (S4-S6), and in numerous illustrations provided in such works as Cundall and Irish (2008:pg 404, fig. 2.25). Perhaps we are referring to different notches, but as mentioned, we illustrate what we mean by this.
Page 6, line 22: "premaxilla's nasal process expands both dorsoventrally and anteroposteriorly". I believe this is incorrect, it expands mediolaterally and anteroposteriorly. It is quite clear that the relative height of the nasal process remains the same if compared to septomaxilla, nasal, and prefrontal.
Disagreed. We made an absolute statement about the nasal process, through the ontogenetic stages we observed, becoming longer and deeper. The Reviewer is attempting to diminish this simple observation by taking our absolute statement of the nasal process in exclusion of all other elements and making it a relative statement by comparing it to other snout elements, something we did not do. This is a trivial distinction and represents the Reviewer's approach to describing anatomy and ontogeny and not our own. We therefore have made no changes to our straightforward observation.
We have changed this sentence to: "Our observations of suspensorium development in T. radix also enable novel interpretations of paedomorphosis in the evolution and ontogeny of the scolecophidian skull." (Our response to the comment on page 19, line 33, as well as our updated section 4.2, fully address the comments made here by the reviewer.) Page 25, line 27 and following: "Furthermore, the absence of a crista circumfenestralis in T. radix […] provides evidence for paedomorphosis in the evolution of this trait." As discussed above, the absence of a crista circumfenestralis in embryonic and adult Thamnophis does not demonstrate that paedomorphosis is the cause of this absence in the latter. Thamnophis alone does not tell us anything about which evolutionary developmental process is responsible for the absence of the CCF in adult snakes. The authors first need to verify that absence of a CCF is a plesiomorphic condition in embryonic or juvenile snakes, if that is the case, then they can conclude that its absence in adult Thamnophis can be interpreted as paedomorphic.
See our response and associated revisions for the comments regarding page 21, line 10.
Page 33, line 6: as discussed above, in booids the quadrate does not always remain upright (see comment on page page 17, line 47) We have changed this sentence to: "(A) Booids (boas, pythons, and relatives) achieve macrostomy via elongation of the supratemporal and quadrate, with the quadrate becoming ventrolaterally deflected but remaining in the same transverse plane." Reference 25 is likely Kamal (1969) (and not 19696).

Changed to: "Frontals completely sutured"
Parietal & Embryo: "Almost entirely unfused along sagittal suture." There is no sagittal suture to speak of, only an unossified central region of the parietal.

Done.
Dentary & Embryo/Juvenile/Adult: To my knowledge, tooth counts on marginal dentigerous elements (dentary and maxilla) are very conservative in snakes within a given species and throughout ontogeny. Please recheck tooth positions, and make sure you don't simply count visible teeth, but tooth positions. Tooth counts from multiple specimens (and from both sides of the skull) would be welcome to make sure that there is no intraspecific variability added to what may vary ontogenetically. See our response to the comment on page 9, line 22. Figure 6: I wonder if showing the angle between quadrate and compound in a tilted perspective of the skull is appropriate (the angles appear smaller than they actually are). I think that showing these angles in lateral view would be much more effective.
We used a tilted perspective for this figure to provide a more direct view of the suspensorium articulation than is visible in lateral or dorsal view. To more accurately display the change in angle of the quadrate, we have moved the angles to Fig. 3, and have added the following sentence to the Fig. 3 caption: "Note the posterior rotation of the quadrate from the juvenile to adult stages." The figure caption contains a sentence that I find misleading: "this pathway of development is unique to T. radix and one other, unrelated genus within the Caenophidia, indicating independent evolution of this pattern". The sentence assumes that only Thamnophis and another caenophidian have a type C macrostomy (see fig. 7), while in fact those are simply the only two snakes where presence of this type of jaw suspension has been noted so far in the literature. There has been no systematic review of jaw suspension in all snakes, therefore making such generalizations is unjustified. The authors should rephrase by writing that "this pathway of development has so far been observed only in …" (do not write that it is unique to two species, we simply do not know that). See our response to the aforementioned comment.

Reviewer: 2
Comments to the Author(s) This is a well-written paper and was a pleasure to read! I rarely review manuscripts that do not require many grammatical and stylistic changes, so, thank you.
Here are my comments: Materials and Methods section: Briefly explain why you only scanned 8 specimens of this widely available species (cost, local availability?). Were the two juveniles from the same mother or were they unrelated? It is not fatal to your conclusions if the juveniles are related, but it would be good to mention.
We've expanded the first paragraph of the Materials and Methods section to address these points.
Regarding relatedness among sampled individuals, we added the following explanation: "Specific information regarding the parentage of the embryonic and juvenile snakes was unavailable. However, given that all of the embryos were accessioned under the same specimen number, it is likely that they are from the same mother. The same line of reasoning also applies to the two juvenile snakes."