Cellular structure of dinosaur scales reveals retention of reptile-type skin during the evolutionary transition to feathers

Domain: earth_science
Published: 2024-05-21
Editorial outcome: minor_revision

Abstract
Fossil feathers have transformed our understanding of integumentary evolution in vertebrates. The evolution of feathers is associated with novel skin ultrastructures, but the fossil record of these changes is poor and thus the critical transition from scaled to feathered skin is poorly understood. Here we shed light on this issue using preserved skin in the non-avian feathered dinosaur Psittacosaurus. Skin in the non-feathered, scaled torso is three-dimensionally replicated in silica and preserves epidermal layers, corneocytes and melanosomes. The morphology of the preserved stratum corneum is consistent with an original composition rich in corneous beta proteins, rather than (alpha-)keratins as in the feathered skin of birds. The stratum corneum is relatively thin in the ventral torso compared to extant quadrupedal reptiles, reflecting a reduced demand for mechanical protection in an elevated bipedal stance. The distribution of the melanosomes in the fossil skin is consistent with melanin-based colouration in extant crocodilians. Collectively, the fossil evidence supports partitioning of skin development in Psittacosaurus: a reptile-type condition in non-feathered regions and an avian-like condition in feathered regions. Retention of reptile-type skin in non-feathered regions would have ensured essential skin functions during the early, experimental stages of feather evolution.

Introduction
Avian feathers fulfil many important roles, most importantly in flight, swimming, insulation, display, sensory function and protection against parasites1. As a result, feathers have long been regarded as the key innovation responsible for the emergence of flight capability in, and adaptive radiations of, birds1,2. Fossilised feathers and feather-like filamentous integumentary structures, however, have been reported in other groups, including non-avian theropods, ornithischians and pterosaurs2–4. Subsequently the term feather has been equivocal in the literature, referring to (1) structures comparable to modern feathers (e.g. ref. 5), (2) the filamentous and pennaceous integumentary structures of theropod dinosaurs (e.g. ref. 6), (3) the pennaceous integumentary structures of avemetatarsalians (e.g. ref. 7) and (4) the filamentous and pennaceous integumentary structures of avemetatarsalians (e.g. ref. 4). Here, we follow the last approach, which defines feather in its broadest sense.

Despite decades of research, the evolutionary origins of feathers remain poorly resolved and, as a result, are the subject of ongoing debate. Certain studies favour independent evolution of feathers in theropods, ornithischians and pterosaurs8,9, but given the shared morphology and histology of the feather structures, and the likely shared genomic heritage and shared pattern of developmental stages of these organisms, we consider a single point of origin more likely2–4,10. At the very least, the available fossil and developmental evidence strongly suggests that very similar, if not identical, genetic and developmental processes underpin the production of feathers in different archosaurian groups2,11,12, regardless of whether a single origin applies.

In extant birds, feathers are associated with complex adaptations of the skin, including (1) a thin, pliable epidermis to facilitate motion during flight13, (2) follicles for the generation and renewal of feathers14, (3) a shift in the site of epidermal melanogenesis to feather follicles for plumage colouration15, (4) a dermal muscular system for support and control of feathers16,17 and (5) a lipid-rich corneous layer for regulation of water- and heat loss18. Avian skin is therefore distinct in anatomy and function to scaled reptilian skin, which presumably represents the ancestral condition14. Little is known, however, about this evolutionary transition, especially the timing and pattern of acquisition of feather-associated skin modifications19. To date, only two studies have examined the skin ultrastructure of basal birds and their close maniraptoran relatives19,20. The feathered skin of these taxa had already acquired certain modern characters, including layers of keratin-rich corneocytes that were shed continuously19 and a dermal system of muscles and connective tissues associated with flight feathers20. Resolution of the early evolution of feather-associated skin traits therefore requires analysis of preserved skin in dinosaurs from earlier-diverging clades. Specimens of these taxa are known to preserve scales and non-scaled skin9,21, but there are few microscopic studies of the preserved remains (but see refs. 22,23).

Here we report ultrastructural preservation of scaled skin from non-feathered body regions of a specimen of Psittacosaurus from the Early Cretaceous Jehol Biota of China. The skin is replicated in three dimensions in silica and preserves evidence of epidermal layers, corneocytes and melanosomes. These ultrastructural details indicate that the non-feathered skin of Psittacosaurus exhibits the plesiomorphic reptilian condition, demonstrating that early evolution of avian skin traits was restricted to feathered body regions.

Results
NJUES-10 (School of Earth Sciences and Engineering, Nanjing University, China) is a juvenile specimen of Psittacosaurus (see Supplementary Note 1 and 2 for taxonomic assignment and ontogenetic status, respectively, of the specimen), a ceratopsian dinosaur known to possess bristle-like monofilaments on the dorsal tail and scales in non-feathered body regions21,24,25. The specimen belongs to the Early Cretaceous (Valanginian–Aptian, ca. 135–120 Ma26) Jehol Biota of northeastern China. It preserves a 664 mm long, near-complete, well-articulated skeleton with its ventral side facing upward (Fig. 1 and Supplementary Fig. 1); a well-defined cluster of gastroliths is preserved in the abdomen (Supplementary Fig. 2a, b). In natural light, no preserved soft tissues are evident. Under ultraviolet (UV) light, however, patches of mineralised soft tissues are evident in the torso (shoulder, chest and abdominal flank regions) and along the limbs (the humerus, radius/ulna and femur) (Figs. 1–2 and Supplementary Fig. 2). The soft tissues fluoresce with an orange-yellow hue that is distinct from the cyan hue of the bones, the green hue of the glue (used to adhere various fragments of the slab together) and the dark purple sedimentary matrix. The soft tissues exhibit a distinct texture defined by closely spaced polygons, although locally the tissues are poorly preserved and the polygons, less obvious (Fig. 2c, e, g). The soft tissues are interpreted as the preserved remains of scaled skin based on their close morphological resemblance to extant and fossil archosaurian scales21,27 and their extensive distribution on the body (Fig. 1 and Supplementary Fig. 2). The preserved scales are tuberculate (i.e., non-overlapping and non-polarised) and polygonal-to-rounded scales. They are mostly ca. 0.8–1.2 mm wide, interspersed with rare larger scales of ca. 1.5–2 mm wide (Supplementary Fig. 3; see also Supplementary Note 3 for additional description of the scale morphology). The former corresponds to basement scales, which in ceratopsian and most other dinosaurs are tuberculate scales (typically 1–10 mm wide) that cover most of the body surface21,28. The rare large scales likely correspond to feature scales that are individually surrounded by basement scales21,28. The preserved Psittacosaurus skin represents ventral skin as it drapes over the skeleton (Fig. 3d and Supplementary Fig. 2c, e).

Ultrastructure of the fossil skin
Samples of the fossil skin were taken from the thorax and abdominal flank regions (Fig. 2b, e, g). SEM analysis of the fossil surface reveals that the skin remains have an amorphous to fine-grained, slightly uneven texture and comprise multiple layers (Supplementary Fig. 4). Hemispherical depressions ca. 5–25 μm in diameter occur locally and comprise aggregates of cubic to subspherical voids, each ca. 0.5–2 μm wide (Supplementary Figs. 4 and 5). These structures are interpreted as external moulds of pyrite framboids that dissolved during diagenesis29.

Vertical sections of the fossil skin show two major layers (Figs. 3–5). The upper layer is ca. 25–60 μm thick and comprises ca. 10–20 sublayers, each ca. 1.8–2.5 μm thick. Each sublayer is amorphous to nanocrystalline with rare microcrystals up to 7 μm wide (Figs. 3–5 and Supplementary Figs. 6–7). Individual sublayers show undulating upper and lower surfaces in vertical section and typically taper laterally (Figs. 3g–i, 4e, h and 5d–f). Vertical fractures divide the sublayers into fragments, many of which persist laterally for at least 20 μm; rare examples persist for at least 50 μm (Figs. 3e–g, 4d, 5d and Supplementary Fig. 4d). The fragments are often lined by isopachous cement comprising fibrous nanocrystals that are orientated orthogonal to the sublayer surfaces (Fig. 4g and Supplementary Fig. 6f). The total thickness of the upper skin layer and the number of constituent sublayers vary among and within samples. This may reflect the loss of some layers during specimen preparation. There is, however, evidence that some of this variation is original: in certain regions from the thorax where the skin is covered by sediment, only ca. 10 sublayers (combined thickness ca. 25 μm) are present (Fig. 5), while more sublayers (combined thickness ca. 60 μm) are clearly present in the abdominal flank regions (Fig. 3).

This upper skin layer strongly resembles the outermost layer of the epidermis in extant reptiles, i.e., the stratum corneum. In extant reptiles, this skin layer varies in thickness (ca. 10–150 μm) among body regions30,31. It comprises a stack of corneocytes—flattened cells typically 15 μm wide that appear elongate and spindle-like in vertical section32,33. These cells fuse laterally during development, forming individual syncytial corneocyte layers with partial (in extant crocodiles, chelonians and tuataras) or complete (in extant squamates) loss of cell boundaries31,32,34,35. The characteristic layering of the reptile stratum corneum is evident in the upper layer of the fossil skin. Many of the sublayer fragments of the fossil skin (Figs. 3e–g, 4d, 5d and Supplementary Fig. 4d) are wider than individual corneocytes in extant reptiles but are consistent with lateral fusing of corneocyte boundaries. Further, corneocyte thickness is highly conserved in modern amniotes, typically 1–3 μm, irrespective of the total skin thickness36. For instance, corneocytes are 1.3–2 μm thick in the crocodile Crocodylus porosus31 and 1.5–2 μm thick in the scutate scales of chicken feet17. These thicknesses are comparable to those of the sublayers in the fossil skin, although the isopachous cement, varying from less than 1 μm to ca. 2 μm thick, adds to the original thickness of the fossil structures. As such, the upper layer of the fossil skin is interpreted as the stratum corneum and each sublayer, as a single layer of (laterally fused) corneocytes. The local lateral tapering of the sublayers likely represents remnant cell boundaries.

The lower layer of the fossil skin is 6–20 μm thick and laterally persistent (Figs. 4–5 and Supplementary Figs. 6–8). It is typically amorphous to nanocrystalline with occasional larger microcrystals up to 11 μm wide (Supplementary Fig. 8c). In extant reptiles, the stratum corneum is underlain by the uncornified, typically thinner inner epidermis, and, in turn, the dermis30,37. Based on its position and thickness relative to the fossil stratum corneum, the lower layer of the fossil skin is therefore interpreted as the uncornified inner epidermis (with possibly the upper part of the dermis).

Fossil skin samples from the thorax region often show striking moulds of oblate to spheroidal microbodies, each ca. 0.2–0.4 μm wide (Figs. 4–5 and Supplementary Figs. 6–9). The distribution of the microbodies varies: they can occur in the lower skin layer only (Figs. 4–5 and Supplementary Figs. 6 and 8) or in both the upper and lower layers (Supplementary Fig. 7), or they can be absent (Supplementary Fig. 6d). These microbodies fall within the size range of fossil and extant melanosomes38 and resemble the low aspect ratio melanosomes reported previously in the integumentary structures of pterosaurs3,38, non-maniraptoran dinosaurs (including Psittacosaurus)25,38 and extant reptiles39; in fact, mouldic preservation of melanosomes is common in many fossils3,25,38. Critically, the spatial distribution of the microbodies is consistent with that of melanosomes in extant crocodilian scales, where most melanosomes occur in the uncornified epidermis (i.e., in melanocytes) and uppermost dermis (i.e., in melanophores)39. In addition, melanosomes derived from melanocytes are incorporated into corneocytes in the stratum corneum, creating macroscopic colour patterns comprising black spots and/or stripes32,39.

An alternative interpretation is that the microbodies may represent the remains of other skin features, fossil bacteria or abiotic (i.e., taphonomic) artefacts. None of these, however, is likely. Although the microbodies are similar in size to pigment granules in xanthophores in the scales of extant crocodiles30,39, these granules are dermal, not epidermal39. Further, the microbodies do not resemble skin glands in extant reptiles. Reptilian skin glands are multicellular features that are much larger than the fossil microbodies and are restricted to certain body regions40. In crocodiles, the closest relative to dinosaurs among extant reptiles, the skin glands are restricted to mandibular, cloacal and dorsal regions40. Finally, although bacteria preserved as moulds in various minerals (including silica) have been reported in fossils and taphonomic experiments41, the microbodies observed in Psittacosaurus are unlikely to be fossilised decay bacteria. This is because (1) decay bacteria, where derived externally to the carcass, normally overgrow the tissues42, whereas the fossil microbodies are clearly internal structures, and (2) even in the case of internally derived decay bacteria, they usually are much more diverse in size (0.1 μm to millimetres) and shape (spirals, bacilliforms and coccoids) than observed for the fossil microbodies43,44. Lastly, while microscopic voids can be produced abiotically in crocodile scales during decay45, such voids exhibit a much wider range of morphologies (a few microns to tens of microns wide and rounded to irregularly shaped) than those of the mouldic microbodies. Collectively, these observations indicate that the microbodies are most parsimoniously interpreted as fossil melanosomes, preserved as three-dimensional moulds.

Chemistry of the fossil skin
Energy dispersive X-ray spectroscopy (EDS) maps show that the fossil skin is rich in Si and O, but no other elements (Fig. 6), indicating that the tissue is replaced in silica. In contrast, the sedimentary matrix is rich in Si, Al and O, consistent with a composition rich in aluminosilicates; some sedimentary grains are rich in Na, Mg, Ca and Zn. The Psittacosaurus bones and clam shrimp shells (from the same bedding plane as the fossil skin) are rich in Ca and P (Supplementary Figs. 10–11), consistent with their in vivo composition, i.e., calcium phosphate46.

The EDS data are supported by micro-Fourier transform infrared spectroscopy (µ-FTIR), which reveals that IR spectra for the fossil skin are almost identical to that of quartz grains from the sedimentary matrix (Fig. 7 and Supplementary Figs. 12–13). The fossil skin and the quartz grains share three characteristic peaks. The two major peaks at ∽1040 cm–1 and ∽770 cm–1 correspond to asymmetric and symmetric stretching vibrations of Si–O–Si, respectively47. The minor peak at ∽1160 cm–1 likely reflects the presence of other cations that may substitute Si within the Si–O–Si lattice47. These spectra differ strongly from the spectrum of the embedding resin (Fig. 7). The IR spectra of the fossil skin lack peaks for organic compounds, which is not unexpected for mineralised soft tissues48.

Methods
Sampling
The studied Psittacosaurus specimen NJUES-10 belongs to the fossil collections of the School of Earth Sciences and Engineering, Nanjing University, Nanjing, China. Approval for study of NJUES-10, including destructive sampling and export of samples, was received from the school. The specimen was donated to Nanjing University from a private collection in 2021; data on locality and stratigraphy are not available. The specimen is preserved on a single, fragmented slab that has been glued together along the fissures. The specimen was examined for evidence of preserved skin using a Nikon SMZ25 stereomicroscope coupled with a UV light source (wavelength 365 nm). Under UV light, discontinuous patches of soft tissue can be readily distinguished from the bones, sediment and glue via distinct differences in fluorescence colour. Regions of soft tissue lacking obvious contamination by glue were selected for further analysis. Small samples (most 2–5 mm wide) of soft tissue were dissected from the thorax and abdominal flank regions (Fig. 2) using a scalpel and mounted on carbon tape on aluminium stubs; selected samples were later embedded in epoxy resin and polished.

Scanning electron microscopy (SEM)
SEM analyses used a JEOL JSM IT-100 variable pressure (VP)-SEM in the School of Biological, Earth and Environmental Sciences, University College Cork. The SEM was equipped with a backscatter detector and a 30 mm2 EDS detector. Samples were examined at accelerating voltages of 10–20 kV and a working distance of 10 mm. Most samples were uncoated for SEM analyses in VP mode; selected samples were sputter-coated with Au for high-resolution imaging in high-vacuum mode.

Micro-attenuated total refection Fourier-transform infrared (µATR-FTIR) spectroscopy
Infrared reflectance spectra were collected from regions of interest in polished vertical sections of the fossil skin in the School of Biological, Earth and Environmental Sciences, University College Cork. Data collection used a PerkinElmer Spotlight 400i FTIR microscope coupled to a Frontier spectrometer, a dedicated high-resolution ATR imaging accessory and a computer. Collection was via the software Spectrum IMAGE R 1.11.2.0016 and the parameters were set as follows: resolution 16 cm−1, 32 scans per pixel, interferometer speed 1.0 cm/s, scan region 4000 cm−1 to 750 cm−1 and pixel size 1.56 μm. The resulting reflectance maps contain an infrared spectrum for each pixel and can be displayed as transmittance or absorbance. A background spectrum was collected prior to each map to account for signal contribution from the instrument and environment. Raw spectra were processed as follows: atmospheric correction (to compensate for water vapour and CO2 contributions) in Spectrum IMAGE R 1.11.2.0016 and baseline correction for extracted spectra in SpectraGryph v1.2.16.1 (using the default coarseness and offset values in the advanced baseline correction function). The Spectrum IMAGE and SpectraGryph software are available from https://www.perkinelmer.com/product/spotlight-400-s-w-kit-sp400-lx108895 and www.effemm2.de/spectragryph, respectively.

Statistics and reproducibility
Image acquisition of the scanning electron micrographs (Figs. 3d–h, 4c–g, 5c–e, g–p, and the first panel of 6 and Supplementary Figs. 4, 6–8, 9c, f, 10a–b, 11a–b and 12a–b, e, h–i) followed convention in the field. These micrographs were obtained as single, unique, images. Repeated acquisition of images in the same region is not standard procedure as it may lead to beam damage.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In their manuscript entitled “Three-dimensional preservation of skin ultrastructure in a feathered dinosaur”, Zixiao Yang and colleagues describe the microstructure of fossil skin exceptionally preserved in three dimensions in the torso region of a new specimen of the ceratopsian dinosaur Psittacosaurus from the Early Cretaceous Jehol Biota of China. The skin is preserved in silica and includes epidermal layers, corneocytes, and melanosomes, providing unique information on these structures in one of the two feathered ornithischian taxa known to date. The authors reveal that the non-feathered scaly skin of this dinosaur’s torso exhibits: i) the plesiomorphic reptilian condition of having an original composition rich in corneous beta proteins rather than (alpha-) keratins (as in birds); ii) evidence for melanin-based colour patterning consistent with that in the scales of extant crocodilians; iii) a thin stratum corneum reflecting probably a reduced demand for mechanical protection in the elevated stance of a bipedal subadult individual.\nI enjoyed very much reading this contribution on the skin ultrastructure of Psittacosaurus. I am admittedly not an expert in skin microstructure as my expertise lies in the morphology and diversity of the scaly integument in non-avian dinosaurs (including Psittacosaurus, which I studied or am studying). My non-expert eyes can hopefully offer some helpful insights to improve this study. The manuscript is clear, well-organized, and well-illustrated and despite my limited knowledge of this topic, I was able to follow and understand the text entirely. I am, however, unable to evaluate the robustness of the evidence supporting the presence of three-dimensional skin and its composition.\nThat being said, Maria E. McNamara is one of the few experts in this field and I, therefore, fully rely on her interpretation of the sample under study. Besides, the authors provide many evidence to support their claims and it is clear to me that they have analyzed a sample of scaly skin, whose polygonal basement scales are clearly revealed in UV light. Although the results of this paper are not groundbreaking (they are totally expected from an evolutionary point of view), this study is important because it is the first to provide information on the skin ultrastructure of an ornithischian and a feathered dinosaur from an early-diverging clades, one that includes both scales and protofeathers. I have, consequently, only minor suggestions and remarks to offer and I would personally accept this contribution with minor revisions.\nMy main remark lies in the absence of information on the skin colour of the Psittacosaurus specimen NJUES-10 despite the presence of fossil melanosomes. The shape of these melanosomes is known to provide direct information on the colour of the skin and I would like the authors to discuss this aspect in their paper. What colour(s) was (were) present in the torso and limbs of NJUES-10 based on the shape and concentration of the melanosomes in the sample analyzed by the authors? Are there any patterns such as stripes? How does it compare to that of the Frankfurt specimen SMF R 4970 (which shows evidence of countershading) and the two specimens PKUPV1050 and PKUP V1051 sampled by Li et al. (2014)?\nIs it unfortunate that NJUES-10 does not preserve the filamentous structure present in the tail of SMF R 4970. This is, actually, not the first Psittacosaurus specimen preserving skin but no monofilament. Similarly, two exquisitely preserved specimens PKUPV1050 and PKUP V1051 illustrated by Li et al. (2014) also show this condition. The monofilaments are quite thick and appear to be robust in SMF R 4970, which made me wonder whether they may have been only present in some Psittacosaurus individuals (the male or the female) or species (less likely). Could the authors discuss the absence of monofilaments in NJUES-10 based on the taphonomical scenario they discuss in their section “Taphonomy of the fossilised skin”? Is it normal that these structures decay faster than the scaly integument? Any information on this topic would be particularly interesting.\nThe description of the preserved integument in NJUES-10 is also rather limited according to me. Can the authors provide, in the text or the supplementary information, additional information on the skin morphology of this specimen and how it compares to the more extensive skin of SMF R 4970? Can they observe any variation in the morphology and size of the basement scales along the torso and the limbs where the skin is preserved? The shoulder region of the forelimb of SMF R 4970, for instance, includes feature scales whereas a hexagram pattern with triangular scales is present in the posterior portion of the upper limbs. It would, therefore, be interesting to know if NJUES-10 shows a similar pattern or if there were some intraspecific variations in skin morphology in Psittacosaurus.\nThe authors assume that the relatively thin stratum corneum of the Psittacosaurus ventral torso “may reflect a reduced demand for mechanical protection in an elevated stance”, therefore arguing that NJUES-10, though to be around three years old, probably represents a bipedal subadult individual (as a quadrupedal adult would have a much thicker stratum corneum). This information is quite interesting and I would invite the authors to provide this information in the abstract if the word limit allows it.\nOther minor remarks:\n- Could the authors provide, in the result section, information on the length of the specimen and the probable stages of the sediments it was found?\n- The scaly skin of NJUES-10 can be described as having tuberculate (i.e., non-overlapping and non-polarised) and polygonal basement scales. The authors can refer to my paper (Hendrickx et al. 2022) for a definition of these scales in dinosaurs.\n- I do not think that the feathers of Kulindadromeus can be described as simple. They are rather complex, with filaments associated with a basal plate and ribbon-shaped structures (Godefroit et al. 2020).\nI provided a few additional remarks and corrections in the Word file, which I invite the authors to check.\nSincerely,\nChristophe Hendrickx, San Miguel de Tucumán, the 15th of November 2023\nReferences associated with this review\nGodefroit, P., Sinitsa, S. M., Cincotta, A., McNamara, M. E., Reshetova, S. A. & Dhouailly, D. 2020. Integumentary structures in Kulindadromeus zabaikalicus, a basal neornithischian dinosaur from the Jurassic of Siberia. In: Foth, C. & Rauhut, O. W. M. (eds) The Evolution of Feathers: From Their Origin to the Present. Springer International Publishing, Cham, Fascinating Life Sciences, 47–65., doi: 10.1007/978-3-030-27223-4_4.\nHendrickx, C., Bell, P. R., Pittman, M., Milner, A. R. C., Cuesta, E., O’Connor, J., Loewen, M., Currie, P. J., Mateus, O., Kaye, T. G. & Delcourt, R. 2022. Morphology and distribution of scales, dermal ossifications, and other non-feather integumentary structures in non-avialan theropod dinosaurs. Biological Reviews, 97, 960–1004, doi: 10.1111/brv.12829.\nLi, Q., Clarke, J. A., Gao, K.-Q., Zhou, C.-F., Meng, Q., Li, D., D’Alba, L. & Shawkey, M. D. 2014. Melanosome evolution indicates a key physiological shift within feathered dinosaurs. Nature, 507, 350–353, doi: 10.1038/nature12973.", "reviewer_2": "Yang et al. present morphological, histological and elemental evidence for high fidelity preservation of multiple epidermal layers (stratum corneum, lower epidermis) in the ornithischian dinosaur, Psittacosaurus. Firstly, I congratulate the authors on their discovery (something that I’ve been hoping to find for many years!) and the coherent, well-written manuscript. While I am generally in favour of the findings and evidence presented, I do think there are several areas that need further exploration, which will strengthen the current manuscript and make it a suitable publication for Nature Communications. My main points for considering are detailed below, but additional edits/comments can be found in the attached marked-up version of the manuscript.\nFirstly, the authors regard the filamentous structures on the tail of Psittacosaurus as homologous with theropod feathers. This, however, remains controversial, and homology has not been conclusively demonstrated. Stating in the title that it is a 'feathered dinosaur' certainly seems overreaching. The findings are interesting enough and stand on their own without the need to invoke feathers at this point. While the authors can ‘side’ with whomever they like, I suggest that they also acknowledge the more circumspect publications on this topic in the remainder of the manuscript (e.g. Barrett et al., 2015; Campione et al. 2020).\nLines 118-123: The authors note variation in the thickness of the upper layer. There is well-documented variation in the thickness of stratum corneum across the body in modern reptiles, which the authors are clearly aware of. It would therefore be helpful if you could explicitly state where ‘thin’ vs ‘thick’ stratum corneum appear in the present specimen and whether it conforms to changes in thickness in modern reptiles, or even birds. The variation in birds is naturally correlated to presence/absence of feathers, so this could be elaborated on in the Discussion (see also comment below re lines 251-262).\nLines 172-185: While I have no a priori concerns about the interpretation of microbodies, the figures do not do them justice: I cannot see the ‘spheroidal’ nature of any of them; rather, they show up as hazy black specks in both the figures and supp info. Since these images are the only ‘evidence’, some higher res images are strongly recommended.\nLine 209: I suspect the FTIR analysis was performed in the hope of identifying organic traces. If so, It might be worth mentioning that no peaks could be attributed to stretch vibrations for organic material, which I actually find a little surprising given the apparent fidelity of the skin layers! Something else to discuss perhaps, from a taphonomic perspective, although I understand space is tight in this format. Micron-scale preservation of features seems to be fairly regularly reported for silicified invertebrate fossils, but not, as far as I’m aware, in vertebrates.\nLines 251-262: the authors compare the thickness of stratum corneum in Psittacosaurus to the ‘non-scaled’ skin of birds and the ventral scales of crocodilians. This seems like an odd comparison, given that there is a well-established relationship in birds, which have thinner stratum corneum in non-feathered regions, whereas it is thicker on the scaled legs (see various Stettenheim references for e.g.). Similarly, since the current paper relates to scales of Psittacosaurus, it would seem more appropriate to comment on scales in birds rather than the non-scaled regions. Further consideration will then also need to be made considering that avian scales are non-homologous with reptilian scales. Subsequently, it feels like an ‘apples to oranges’ comparison at present. Secondly, while the comparisons are topographically equivalent between crocs and Psittacosaurus (i.e. ventral scales), it would also be worth commenting on the variation between body regions in crocs. I am unsure how absolute values for stratum corneum thickness vary with ontogeny in modern reptiles and birds, but I encourage the authors to explore and comment on this potential variation as well as it has strong bearing on the current paper (i.e. a 3-6m long C. porosus may not be a suitable comparison with a juvenile Psittacosaurus!). Considering both of these aspects would greatly strengthen their argument. The conclusion on line 262 that “the original skin thickness to have fallen within the range of thicknesses exhibited by extant crocodilians and not birds” therefore needs further consideration. An additional sentence comparing your values to those reported in hadrosaurs by Fabbri et al (2019) and Barbi et al (2019) would also be relevant here. The latter paper was not cited, but should be (see also line 49).\nBarbi, M., Bell, P.R., Fanti, F., Dynes, J.J., Kolaceke, A., Buttigieg, J., Coulson, I.M. and Currie, P.J., 2019. Integumentary structure and composition in an exceptionally well-preserved hadrosaur (Dinosauria: Ornithischia). PeerJ, 7, p.e7875.\nLines 341-451: The authors state that the specimen had not undergone “bloat and float” based on the articulation of element and the intact mass of gastroliths. While I agree that the specimen did not remain exposed and likely hadn’t been dead for long, it is difficult to imagine how a terrestrial dinosaur could end up within a fine-grained, presumably deep lacustrine environment had it not floated out to it final resting place. A bloat and float scenario would seem a better explanation for why we have an animal (1) preserved belly-up (2) in a quiet, deep water setting. While I agree the degree of articulation may appear at odds with the findings of, say, Syme and Salisbury (2014), the other evidence is more compelling in this case. Perhaps of interest, the overall proportions of a juvenile Psittacosaurus (especially this one) do recall the proportions of ankylosaurs, which are frequently overturned, presumably also as a result of bloat and float (see Mallon et al. 2018 Pal. Pal. Pal.). One other question pertinent to this argument is: is the specimen actually preserved belly up, or has it just been prepared that way? Although the provenance of the specimen is unknown, there should (hopefully) be some sedimentary markers that indicate way up. This did not come across clearly in the results, which simply state “It preserves a near-complete, well-articulated skeleton in ventral aspect”. The orientation should be backed-up and confirmed as it is relevant to the taphonomy of the specimen. For the record, I have no issue with the other aspects of their taphonomic interpretation.\nLines 398-399: The authors state that Psittacosaurs “clearly exhibits the reptilian condition”, but as mentioned, there needs to be a more detailed comparison to the scales of birds before this statement can be considered.\nI hope these comments are helpful.\nRegards,\nPhil Bell"}, "author_response": "Response to reviewers\nWe sincerely thank all the reviewers for their insightful and constructive comments\nand suggestions on our manuscript. Following the reviewers’ comments and\nsuggestions, we have carefully revised the manuscript. We have also addressed all the\ncomments and questions, which are detailed below with our responses in red.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nComment 1: In their manuscript entitled “Three-dimensional preservation of skin\nultrastructure in a feathered dinosaur”, Zixiao Yang and colleagues describe the microstructure\nof fossil skin exceptionally preserved in three dimensions in the torso region of a new\nspecimen of the ceratopsian dinosaur Psittacosaurus from the Early Cretaceous Jehol Biota of\nChina. The skin is preserved in silica and includes epidermal layers, corneocytes, and\nmelanosomes, providing unique information on these structures in one of the two feathered\nornithischian taxa known to date. The authors reveal that the non-feathered scaly skin of this\ndinosaur’s torso exhibits: i) the plesiomorphic reptilian condition of having an original\ncomposition rich in corneous beta proteins rather than (alpha-) keratins (as in birds); ii)\nevidence for melanin-based colour patterning consistent with that in the scales of extant\ncrocodilians; iii) a thin stratum corneum reflecting probably a reduced demand for mechanical\nprotection in the elevated stance of a bipedal subadult individual.\nI enjoyed very much reading this contribution on the skin ultrastructure of Psittacosaurus. I\nam admittedly not an expert in skin microstructure as my expertise lies in the morphology and\ndiversity of the scaly integument in non-avian dinosaurs (including Psittacosaurus, which I\nstudied or am studying). My non-expert eyes can hopefully offer some helpful insights to\nimprove this study. The manuscript is clear, well-organized, and well-illustrated and despite\nmy limited knowledge of this topic, I was able to follow and understand the text entirely. I am,\nhowever, unable to evaluate the robustness of the evidence supporting the presence of three-\ndimensional skin and its composition. That being said, Maria E. McNamara is one of the few\nexperts in this field and I, therefore, fully rely on her interpretation of the sample under study.\nBesides, the authors provide many evidence to support their claims and it is clear to me that\nthey have analyzed a sample of scaly skin, whose polygonal basement scales are clearly\nrevealed in UV light. Although the results of this paper are not groundbreaking (they are totally\nexpected from an evolutionary point of view), this study is important because it is the first to\nprovide information on the skin ultrastructure of an ornithischian and a feathered dinosaur\nfrom an early-diverging clades, one that includes both scales and protofeathers. I have,\nconsequently, only minor suggestions and remarks to offer and I would personally accept this\ncontribution with minor revisions.\nResponse: Many thanks for your appreciation of our work and your critical comments. We have\nmade the suggested revisions and responded in detail to each point below. Please note that\nthe line numbers in the red text correspond to the clean version of the revised manuscript.\nComment 2: My main remark lies in the absence of information on the skin colour of the\nPsittacosaurus specimen NJUES-10 despite the presence of fossil melanosomes. The shape of\nthese melanosomes is known to provide direct information on the colour of the skin and I\nwould like the authors to discuss this aspect in their paper. What colour(s) was (were) present\nin the torso and limbs of NJUES-10 based on the shape and concentration of the melanosomes\nin the sample analyzed by the authors? Are there any patterns such as stripes? How does it\ncompare to that of the Frankfurt specimen SMF R 4970 (which shows evidence of countershading)\nand the two specimens PKUPV1050 and PKUP V1051 sampled by Li et al. (2014)?\nResponse: We thank the reviewer for these intriguing questions. A correlation between\nmelanosome geometry and colour has been established only for mammalian hair and\nmaniraptoran feathers (Li et al. 2014). According to the data published in that study, there is\nno significant correlation between melanosome geometry and coloration in reptilian scales.\nWe can thus comment only in very broad terms regarding the coloration of the Psittacosaurus\nintegument. The observed lateral variation in melanosome distribution in the skin suggests at\nleast millimetre-scale spatial variation in skin tone. However, the small size of the samples\nanalyzed renders it impossible to comment on the granularity of that variation in tone, or the\nextent of the variation in tone across the body. We can, however, say that the variation in\nmelanosome abundance is consistent with colour patterning but does not exclude the\npossibility that variations in tone were highly localised.\nWe have added the discussion on the skin colour of NJUES-10 and comparison with other\nspecimens in the section “Skin colour of Psittacosaurus” in Supplementary Information. We\nhave also added a new figure (Supplementary Fig. 9) showing the spatial extent of possible\nskin tone variation.\nReference\nLi, Q. et al. Melanosome evolution indicates a key physiological shift within feathered\ndinosaurs. Nature 507, 350–353 (2014).\nComment 3: Is it unfortunate that NJUES-10 does not preserve the filamentous structure\npresent in the tail of SMF R 4970. This is, actually, not the first Psittacosaurus specimen\npreserving skin but no monofilament. Similarly, two exquisitely preserved specimens\nPKUPV1050 and PKUP V1051 illustrated by Li et al. (2014) also show this condition. The\nmonofilaments are quite thick and appear to be robust in SMF R 4970, which made me wonder\nwhether they may have been only present in some Psittacosaurus individuals (the male or the\nfemale) or species (less likely). Could the authors discuss the absence of monofilaments in\nNJUES-10 based on the taphonomical scenario they discuss in their section “Taphonomy of\nthe fossilised skin”? Is it normal that these structures decay faster than the scaly integument?\nAny information on this topic would be particularly interesting.\nResponse: Agreed. We thank the reviewer for the suggestion and we have added discussion in\nthe section “The lack of feather preservation in NJUES-10” in the Supplementary Information.\nWe did not include this discussion in the main text because we feel the main focus of the study\nis on the preserved skin rather than the lack of feathers. Regarding the lack of preserved\nfilaments: we feel that this may reflect the mode of preservation of the soft tissues in this\nspecimen. Almost all fossil feathers are preserved as organic (carbonaceous) remains. There\nare no known examples of feathers preserved as three-dimensional authigenic replacements\nin silica (as per the skin). It is possible that feathers have an extremely low likelihood of\npreserving in this way.\nComment 4: The description of the preserved integument in NJUES-10 is also rather limited\naccording to me. Can the authors provide, in the text or the supplementary information,\nadditional information on the skin morphology of this specimen and how it compares to the\nmore extensive skin of SMF R 4970? Can they observe any variation in the morphology and\nsize of the basement scales along the torso and the limbs where the skin is preserved? The\nshoulder region of the forelimb of SMF R 4970, for instance, includes feature scales whereas a\nhexagram pattern with triangular scales is present in the posterior portion of the upper limbs.\nIt would, therefore, be interesting to know if NJUES-10 shows a similar pattern or if there were\nsome intraspecific variations in skin morphology in Psittacosaurus.\nResponse: Agreed. We have provided additional description of the preserved scales in both\nthe main text (Results section, lines 93–100) and the Supplementary Information (section\n“Variation in preserved scale morphology in NJUES-10” and Supplementary Fig. 3).\nComment 5: The authors assume that the relatively thin stratum corneum of the\nPsittacosaurus ventral torso “may reflect a reduced demand for mechanical protection in an\nelevated stance”, therefore arguing that NJUES-10, though to be around three years old,\nprobably represents a bipedal subadult individual (as a quadrupedal adult would have a much\nthicker stratum corneum). This information is quite interesting and I would invite the authors\nto provide this information in the abstract if the word limit allows it.\nResponse: Agreed. We have added this information in the abstract (lines 18–20).\nOther minor remarks:\nComment 6: Could the authors provide, in the result section, information on the length of the\nspecimen and the probable stages of the sediments it was found?\nResponse: Agreed. We have provided the information in the Results section (lines 77–80).\nComment 7: The scaly skin of NJUES-10 can be described as having tuberculate (i.e., non-\noverlapping and non-polarised) and polygonal basement scales. The authors can refer to my\npaper (Hendrickx et al. 2022) for a definition of these scales in dinosaurs.\nResponse: Agreed. We have revised accordingly in the Results section (lines 93–94) and in the\nSupplementary information.\nComment 8: I do not think that the feathers of Kulindadromeus can be described as simple.\nThey are rather complex, with filaments associated with a basal plate and ribbon-shaped\nstructures (Godefroit et al. 2020).\nResponse: Agreed. The feathers of Kulindadromeus are now described as filamentous instead\nof simple (line 480).\nI provided a few additional remarks and corrections in the Word file, which I invite the authors\nto check.\nComments in the attached document:\nComment 9: (Line 18) I would suggest to add the fact that this is an avian trait, because most\npeople are unaware of this.\nResponse: Agreed and we have revised accordingly (line 18).\nComment 10: (Line 63) Please provide the stages if possible.\nResponse: Agreed. We have provided the stages in the revised manuscript (line 78).\nComment 11: (Lines 64–65) Please provide information on the length of the specimen.\nResponse: Agreed. We have provided the length in the revised manuscript (line 79–80).\nComment 12: (Line 68) Please be more specific. What limb, what part of the torso?\nResponse: Agreed. The soft tissues are evident in the trunk (shoulder, chest and abdominal\nflank regions) and along the limbs (arms and femurs). We have revised accordingly (lines 83–\n85).\nComment 13: (Line 78) You can also cite my paper, cited in [81].\nResponse: Agreed and we have revised accordingly (line 99).\nComment 14: (Line 79) Do you note any variation in the size and morphology the basement\nscales? Have you noticed feature scales, typically present in the upper limb of Psittacosaurus?\nResponse: The preserved basement scales vary in size and morphology across the specimen.\nThe preserved features scales are rare and occur on the flank of the lower abdomen. We have\nprovided additional description of the preserved scales in both the main text (Results section,\nlines 93–100) and the Supplementary Information (section “Variation in preserved scale\nmorphology in NJUES-10” and Supplementary Fig. 3).\nComment 15: (Line 80) Does it apply to the limbs as well? Is it the ventral skin, the one from\nthe limbs?\nResponse: Yes, the preserved skin layer drapes over the limbs as well. We have revised the\nResults section (line 100–102) and Supplementary Fig. 2c and e to make this clear.\nComment 16: (Line 92) Please specify in here from what part of the body the skin sample was\ntaken from.\nResponse: Agreed and we have revised accordingly (lines 114–115).\nComment 17: (Lines 176–186) Because you assume the presence of fossil melanosomes in this\npart of the body, isn’t there anything to say about the putative colour of this body part of\nPsittacosaurus? Can you perhaps talk a bit more about their shape and concentration, and\nwhat it tell us about the colour of the skin?\nResponse: This comment is effectively the same as Comment 2. Please see our response to\nComment 2.\nComment 18: (Lines 301–304) This is quite interesting and I would suggest to provide this\nresult in the abstract.\nResponse: Agreed and we have revised accordingly (lines 18–20).\nComment 19: (Lines 376–377) What about the filamentous structures?\nResponse: This comment is effectively the same as Comment 3. Please see our response to\nComment 3.\nComment 20: (Line 424) Unlike Juravenator, the feathers of Kulindadromeus are far from\nsimple as they include filaments associated with a basal plate and ribbon-shaped structures\n(Godefroit et al., 2020).\nGodefroit, P. et al. Integumentary structures in Kulindadromeus zabaikalicus, a basal\nneornithischian dinosaur from the Jurassic of Siberia. in The Evolution of Feathers: From\nTheir Origin to the Present (eds. Foth, C. & Rauhut, O. W. M.) 47–65 (Springer International\nPublishing, 2020). doi:10.1007/978-3-030-27223-4_4.\nResponse: Agreed. The feathers of Kulindadromeus are now described as filamentous instead\nof simple (line 480).\nComment 21: (Line 434) This has to be specified given that all avemetatarsalian/dinosaurs have\nreticulate scales on their hand/feet. See my Biological review paper (Hendrickx et al., 2022).\nResponse: Agreed and we have revised accordingly (line 477).\nComment 22: (Lines 439–440) This indeed appears to be the case in Juravenator, but not in\npterosaurs, which are currently the earliest branching archosaurs preserving feathers (if they\nare indeed “true” feathers homologous to those of theropods).\nResponse: Agreed. Indeed the condition (of early feathers being sparse and highly localised)\nhas been observed in dinosaurs but not in pterosaurs. This difference may be an artefact of\ndifferent sample sizes (feathers have been reported in only three pterosaur specimens).\nNevertheless we have added this to the Discussion (lines 495–497).\nComment 23: (Lines 460–461) Please specify in here from what precise zone of the bone the\nsamples where taken.\nResponse: Agreed and we have revised accordingly (lines 521–522).\nComment 24: (Line 434) I would suggest to provide information on the place where the\nsamples were examined using a SEM.\nResponse: Agreed and we have revised accordingly (lines 526–527).\nSincerely,\nChristophe Hendrickx, San Miguel de Tucumán, the 15th of November 2023\nReferences associated with this review\nGodefroit, P., Sinitsa, S. M., Cincotta, A., McNamara, M. E., Reshetova, S. A. & Dhouailly, D.\n2020. Integumentary structures in Kulindadromeus zabaikalicus, a basal neornithischian\ndinosaur from the Jurassic of Siberia. In: Foth, C. & Rauhut, O. W. M. (eds) The Evolution of\nFeathers: From Their Origin to the Present. Springer International Publishing, Cham,\nFascinating Life Sciences, 47–65., doi: 10.1007/978-3-030-27223-4_4.\nHendrickx, C., Bell, P. R., Pittman, M., Milner, A. R. C., Cuesta, E., O’Connor, J., Loewen, M.,\nCurrie, P. J., Mateus, O., Kaye, T. G. & Delcourt, R. 2022. Morphology and distribution of scales,\ndermal ossifications, and other non-feather integumentary structures in non-avialan theropod\ndinosaurs. Biological Reviews, 97, 960–1004, doi: 10.1111/brv.12829.\nLi, Q., Clarke, J. A., Gao, K.-Q., Zhou, C.-F., Meng, Q., Li, D., D’Alba, L. & Shawkey, M. D. 2014.\nMelanosome evolution indicates a key physiological shift within feathered dinosaurs. Nature,\n507, 350–353, doi: 10.1038/nature12973.\nReviewer #2 (Remarks to the Author):\nComment 1: Yang et al. present morphological, histological and elemental evidence for high\nfidelity preservation of multiple epidermal layers (stratum corneum, lower epidermis) in the\nornithischian dinosaur, Psittacosaurus. Firstly, I congratulate the authors on their discovery\n(something that I’ve been hoping to find for many years!) and the coherent, well-written\nmanuscript. While I am generally in favour of the findings and evidence presented, I do think\nthere are several areas that need further exploration, which will strengthen the current\nmanuscript and make it a suitable publication for Nature Communications. My main points for\nconsidering are detailed below, but additional edits/comments can be found in the attached\nmarked-up version of the manuscript.\nResponse: We thank the reviewer for the generous and helpful feedback. We have made the\nsuggested revisions and responded in detail to each point below. Responses to the additional\ncomments (from the marked-up version of the manuscript attached by the reviewer) are at\nthe end of this document. Please note that the line numbers in the red text correspond to the\nclean version of the revised manuscript.\nComment 2: Firstly, the authors regard the filamentous structures on the tail of Psittacosaurus\nas homologous with theropod feathers. This, however, remains controversial, and homology\nhas not been conclusively demonstrated. Stating in the title that it is a 'feathered dinosaur'\ncertainly seems overreaching. The findings are interesting enough and stand on their own\nwithout the need to invoke feathers at this point. While the authors can ‘side’ with whomever\nthey like, I suggest that they also acknowledge the more circumspect publications on this topic\nin the remainder of the manuscript (e.g. Barrett et al., 2015; Campione et al. 2020).\nResponse: The term “feather” has been equivocal in the literature. It has been used to refer to\na variety of integumentary structures, including (1) the feathers of birds, (2) the filamentous\nand pennaceous integumentary structures of theropod dinosaurs, (3) the pennaceous\nintegumentary structures of avemetatarsalians and (4) the filamentous and pennaceous\nintegumentary structures of avemetatarsalians.\nTo avoid confusion, we have clarified our definition of “feather” in the Introduction section\n(lines 30–38) in the revised manuscript. We have followed the approach that defines “feather”\nin its broadest sense, i.e., the filamentous and pennaceous integumentary structures of\navemetatarsalians. We use “group” + “feather” to refer to feathers of specific groups (e.g.\n“avian feathers”, “theropod feathers”, “pterosaur feathers”, etc.). We feel this approach is\nappropriate as it does not necessarily imply a single origin. Similarly, the term “scale” has been\nwidely used to refer to both reptilian scales and avian scales that evolved independently.\nWe agree that it is possible that feathers evolved independently in theropods, ornithischians\nand pterosaurs. However, given the shared morphology and histology of these feather\nstructures, and the likely shared genomic heritage and shared pattern of developmental stages\nof these organisms, we believe that a common origin is more likely. At the very least, the\nstriking morphological and histological similarities among the early feathers of the different\ngroups, and the deep homology of extant reptilian scales and avian feathers, strongly suggest\na very similar, if not identical, developmental process underlying the production of these early\nfeathers, regardless of whether they arose from a single origination event or not.\nWe therefore feel that the scales and feathers of Psittacosaurus are extremely relevant to\nunderstanding the early evolution of feathers as a whole, particularly the scale-feather\ntransition, despite the possibility of convergent feather evolution. Critically, our finding of\nreptile-style skin histology in non-feathered regions of Psittacosaurus, and the previous finding\nof tail feathers, are two key pieces of evidence that collectively suggest that the acquisition of\nskin modifications during the scale-feather transition may initially have been localised to\nfeathered regions.\nAs such, we think it is critical to refer to feathers early in the paper, and that it is appropriate\nto refer to Psittacosaurus as a feathered dinosaur. We acknowledge, however, controversy\nregarding the homology of the Psittacosaurus feathers with theropod feathers and have\nrevised the Introduction (lines 39–47) and Discussion (the section “Evolutionary implications”,\nlines 497–500) accordingly.\nComment 3: Lines 118-123: The authors note variation in the thickness of the upper layer.\nThere is well-documented variation in the thickness of stratum corneum across the body in\nmodern reptiles, which the authors are clearly aware of. It would therefore be helpful if you\ncould explicitly state where ‘thin’ vs ‘thick’ stratum corneum appear in the present specimen\nand whether it conforms to changes in thickness in modern reptiles, or even birds. The\nvariation in birds is naturally correlated to presence/absence of feathers, so this could be\nelaborated on in the Discussion (see also comment below re lines 251-262).\nResponse: Agreed. We have clarified the body regions where the thickness of stratum corneum\nvaries (lines 144–148). We have also added comparison of this thickness variation across the\nbody with extant crocodiles (lines 325–329 in the Discussion section “Anatomy of non-\nfeathered skin in Psittacosaurus”) but not birds, because the former is a better extant analogue\nin terms of anatomy (having scales in body regions equivalent to those of Psittacosaurus) and\necology (having a terrestrial rather than volant lifestyle).\nComment 4: Lines 251-262: the authors compare the thickness of stratum corneum in\nPsittacosaurus to the ‘non-scaled’ skin of birds and the ventral scales of crocodilians. This\nseems like an odd comparison, given that there is a well-established relationship in birds,\nwhich have thinner stratum corneum in non-feathered regions, whereas it is thicker on the\nscaled legs (see various Stettenheim references for e.g.). Similarly, since the current paper\nrelates to scales of Psittacosaurus, it would seem more appropriate to comment on scales in\nbirds rather than the non-scaled regions. Further consideration will then also need to be made\nconsidering that avian scales are non-homologous with reptilian scales. Subsequently, it feels\nlike an ‘apples to oranges’ comparison at present. Secondly, while the comparisons are\ntopographically equivalent between crocs and Psittacosaurus (i.e. ventral scales), it would also\nbe worth commenting on the variation between body regions in crocs. I am unsure how\nabsolute values for stratum corneum thickness vary with ontogeny in modern reptiles and\nbirds, but I encourage the authors to explore and comment on this potential variation as well\nas it has strong bearing on the current paper (i.e. a 3-6m long C. porosus may not be a suitable\ncomparison with a juvenile Psittacosaurus!). Considering both of these aspects would greatly\nstrengthen their argument. The conclusion on line 262 that “the original skin thickness to have\nfallen within the range of thicknesses exhibited by extant crocodilians and not birds” therefore\nneeds further consideration. An additional sentence comparing your values to those reported\nin hadrosaurs by Fabbri et al (2019) and Barbi et al (2019) would also be relevant here. The\nlatter paper was not cited, but should be (see also line 49).\nBarbi, M., Bell, P.R., Fanti, F., Dynes, J.J., Kolaceke, A., Buttigieg, J., Coulson, I.M. and Currie, P.J.,\n2019. Integumentary structure and composition in an exceptionally well-preserved hadrosaur\n(Dinosauria: Ornithischia). PeerJ, 7, p.e7875.\nResponse: Agreed and we have revised the manuscript accordingly. Specifically, we have added\nanatomical comparisons of the stratum corneum of Psittacosaurus with that of (1) crocodilian\nscales (regarding variation in body regions and ontogeny; lines 304–307, 325–329), (2) avian\nscutate and reticulate scales (lines 307–309, 335–338) and (3) the reported hadrosaur scales\n(lines 294–300, 309–311). These revisions are provided in the Discussion section “Anatomy of\nnon-feathered skin in Psittacosaurus”.\nComment 5: Lines 398-399: The authors state that Psittacosaurs “clearly exhibits the reptilian\ncondition”, but as mentioned, there needs to be a more detailed comparison to the scales of\nbirds before this statement can be considered.\nResponse: Palaeontological and developmental evidence supports the hypothesis that avian\nscales are secondarily derived from feathers and are thus non-homologous with reptilian\nscales, as also noted by the reviewer (in Comment 4). As such, avian scales are not relevant in\nterms of the evolutionary transition from scales to feathers. We therefore maintain that it is\nappropriate to compare Psittacosaurus skin with reptilian scales (the pleiomorphic condition)\nand avian feathered skin (the derived condition) when addressing the scale-feather transition.\nWe have revised the Discussion section “Evolutionary implications” (lines 449–456) to make\nour reasoning clearer.\nComment 6: Lines 172-185: While I have no a priori concerns about the interpretation of\nmicrobodies, the figures do not do them justice: I cannot see the ‘spheroidal’ nature of any of\nthem; rather, they show up as hazy black specks in both the figures and supp info. Since these\nimages are the only ‘evidence’, some higher res images are strongly recommended.\nResponse: We thank the reviewer for the suggestion of additional higher-resolution images of\nthe mouldic melanosomes. We have attempted TEM sectioning on multiple fossil skin samples,\nbut unfortunately the preservation in silica does not facilitate the preparation of high-quality\nTEM sections as the fossil skin is too friable. We think however that the presented images are\nsufficient to support our conclusion, because (1) the interpretation of the microbodies as\nmelanosomes is not disputed by any of the reviewers and (2) we relied on the\npresence/absence, not the shape, of the fossil melanosomes to infer colour patterns.\nComment 7: Line 209: I suspect the FTIR analysis was performed in the hope of identifying\norganic traces. If so, It might be worth mentioning that no peaks could be attributed to stretch\nvibrations for organic material, which I actually find a little surprising given the apparent\nfidelity of the skin layers! Something else to discuss perhaps, from a taphonomic perspective,\nalthough I understand space is tight in this format. Micron-scale preservation of features\nseems to be fairly regularly reported for silicified invertebrate fossils, but not, as far as I’m\naware, in vertebrates.\nResponse: We did indeed test the fossil skin for evidence of organic compounds using FTIR\nanalysis. Given the mode of preservation of the fossil skin (3D authigenic replacement in silica),\nhowever, the likelihood of recovering organic moieties is low (Briggs 2003). Nevertheless we\nhave added a statement regarding the absence of organic peaks in the Results section (lines\n259–260) of the revised manuscript.\nReference\nBriggs, D.E. The role of decay and mineralization in the preservation of soft-bodied fossils.\nAnnu. Rev. Earth Planet. Sci. 31, 275–301 (2003).\nComment 8: Lines 341-451: The authors state that the specimen had not undergone “bloat\nand float” based on the articulation of element and the intact mass of gastroliths. While I agree\nthat the specimen did not remain exposed and likely hadn’t been dead for long, it is difficult\nto imagine how a terrestrial dinosaur could end up within a fine-grained, presumably deep\nlacustrine environment had it not floated out to it final resting place. A bloat and float scenario\nwould seem a better explanation for why we have an animal (1) preserved belly-up (2) in a\nquiet, deep water setting. While I agree the degree of articulation may appear at odds with\nthe findings of, say, Syme and Salisbury (2014), the other evidence is more compelling in this\ncase. Perhaps of interest, the overall proportions of a juvenile Psittacosaurus (especially this\none) do recall the proportions of ankylosaurs, which are frequently overturned, presumably\nalso as a result of bloat and float (see Mallon et al. 2018 Pal. Pal. Pal.). One other question\npertinent to this argument is: is the specimen actually preserved belly up, or has it just been\nprepared that way? Although the provenance of the specimen is unknown, there should\n(hopefully) be some sedimentary markers that indicate way up. This did not come across\nclearly in the results, which simply state “It preserves a near-complete, well-articulated\nskeleton in ventral aspect”. The orientation should be backed-up and confirmed as it is relevant\nto the taphonomy of the specimen. For the record, I have no issue with the other aspects of\ntheir taphonomic interpretation.\nResponse: The specimen is indeed preserved ventral surface uppermost based on soft-\nsediment deformation structures caused by the impacting carcass at deposition and normal\ngrading in the matrix sediment – we have revised the Results section (lines 79–80) and\nprovided additional images of the sedimentary structures (Supplementary Fig. 1) to make this\nclear.\nWe are not entirely certain in what sense the reviewer refers to “bloat and float”. In much of\nthe taphonomic literature, this phrase refers to the (partial) refloating of carcasses after\ndeposition due to the build-up of decay gases. However the reviewer seems to use a different\nmeaning, i.e. the floating of a carcass during transport and prior to deposition. In either case,\nwe feel that any period of extended flotation at or near the water surface likely would have\nresulted in much more extensive disarticulation than observed in NJUES-10. Further, many (if\nnot most) of the terrestrial vertebrates from the Jehol Biota are well-articulated and preserved\nin fine-grained lacustrine deposits (Xu et al. 2020). This contrasts with the extensive\ndisarticulation of the carcasses frequently associated with the “bloat and float” stage in\ntaphonomic experiments (as noted also by the reviewer).\nTherefore, we feel it is more likely that carcasses of terrestrial animals were transported to the\ncentral parts of the Jehol lake(s) via surface currents arising from fluvial input and did not go\nthrough a “bloat and float” stage. The belly-up posture of NJUES-10 could have been\naccidental: the overall rounded shape of the body in vertical section would have rendered the\nanimal unstable in the water column. Indeed, Psittacosaurus preserved with the dorsal side\nup is also known from the Jehol Biota (e.g. Jiang et al. 2014).\nReferences\nJiang, B., Harlow, G.E., Wohletz, K., Zhou, Z. & Meng, J. New evidence suggests pyroclastic\nflows are responsible for the remarkable preservation of the Jehol biota. Nat. Commun. 5,\n3151 (2014).\nXu, X., Zhou, Z., Wang, Y. and Wang, M. Study on the Jehol Biota: recent advances and future\nprospects. Sci. China Earth Sci. 63, 757–773 (2020).\nI hope these comments are helpful.\nRegards,\nPhil Bell\nComments in the attached document:\nComment 9: (Line 30) Highly contentious. I suggest modifying: \"possibly even dinosaurs\".\nResponse: Agreed. We have revised the Introduction (lines 39–47) to acknowledge the\ncontroversy regarding feather origins.\nComment 10: (Line 49) Consider adding Barbi et al 2019, who also describe the stratum\ncorneum in a hadrosaur:\nBarbi, M., Bell, P.R., Fanti, F., Dynes, J.J., Kolaceke, A., Buttigieg, J., Coulson, I.M. and Currie, P.J.,\n2019. Integumentary structure and composition in an exceptionally well-preserved hadrosaur\n(Dinosauria: Ornithischia). PeerJ, 7, p.e7875.\nResponse: Agreed and we have revised accordingly (line 64).\nComment 11: (Line 76) This seems fairly straightforward from your decription, but could you\nplease explicitly state whether there is any variation in this pattern on different parts of the\nbody?\nResponse: Agreed. We have provided additional description of the scalation in the Results\nsection (lines 93–100) and Supplementary Information (section “Variation in preserved scale\nmorphology in NJUES-10” and Supplementary Fig. 3).\nComment 12: (Line 104, Fig. 2) Scale bar in (h) overlaps the region on interest. Please move\nthe scale bar so it is visible.\nResponse: Agreed and we have revised Fig.2 accordingly.\nComment 13: (Line 132) does this refer to the regions between vertical fractures e.g. dashed\nline in Fig 2i. Please clarify.\nResponse: Yes, the sublayer fragments refer to the regions between vertical fractures. This was\nclarified in the original manuscript (line 114; line 138 in the revised manuscript); we have\nadded references to specific figures in the revised manuscript (lines 157–158).\nComment 14: (Line 137) scutate?\nResponse: Yes, the dorsal scales refer to the scutate scales; we have revised accordingly (line\n162).\nComment 15: (Line 219, Fig. 4) It would be helpful to mark the skin-sediment boundary in the\nSEM image.\nResponse: Agreed and we have revised Fig. 4 accordingly.\nComment 16: (Line 285) Perhaps define this acronym again here since the definition only\nappears on pg 1.\nResponse: Agreed and we have revised accordingly (line 333).\nComment 17: (Line 288) what about avian scales?\nResponse: We have provided the relevant discussion in the revised manuscript (line 335–338).\nComment 18: (Line 398) Again, avian scales need to be more closely considered here.\nResponse: As in our response to Comment 5, avian scales are not relevant in terms of the\nevolutionary transition from scales to feathers, given that avian scales are secondarily derived\nfrom feathers and non-homologous with reptilian scales. Instead, it is appropriate to compare\nPsittacosaurus skin with reptilian scales (the pleiomorphic condition) and avian feathered skin\n(the derived condition) when addressing the scale-feather transition. We have revised the\nDiscussion section “Evolutionary implications” (lines 449–456) to make our reasoning clearer.\nComment 19: (Line 427) You should differentiate between types of avian skin, since avian\nscales are secondarily derived structures (i.e. derived).\nResponse: This comment is effectively the same as Comment 18. Please see our response to\nComment 18.\nComment 20: (Line 459) Presumably these are the three locations identified in Fig 1. Please\nreference the figure components here.\nResponse: Agreed and we have revised accordingly (lines 520–522)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I am glad to see that the authors have considered all my remarks and suggestions and would, therefore, suggest to accept the revised version of their manuscript for publication. I admittedly did not know about the lack of correlation between melanosome geometry and colour in reptilian scales and I appreciate the fact that this was discussed by the authors in the Supplementary Information. I have no other remarks to provide at this stage and only feel the need to apologize for the numerous English mistakes I left in my review.\nSincerely,\nChristophe Hendrickx, San Miguel de Tucumán, the 14th of March 2024", "reviewer_2": "I thank the authors for their considered responses and careful revision of the MS. I have no further comments and look forward to seeing the final published version soon."}, "author_response": "Response to reviewers\nWe sincerely thank all the reviewers for their insightful and constructive comments\nand suggestions on our manuscript. We are glad that our revisions and responses from\nlast submission are satisfactory to the reviewers.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nI am glad to see that the authors have considered all my remarks and suggestions and would,\ntherefore, suggest to accept the revised version of their manuscript for publication. I\nadmittedly did not know about the lack of correlation between melanosome geometry and\ncolour in reptilian scales and I appreciate the fact that this was discussed by the authors in the\nSupplementary Information. I have no other remarks to provide at this stage and only feel the\nneed to apologize for the numerous English mistakes I left in my review.\nSincerely,\nChristophe Hendrickx, San Miguel de Tucumán, the 14th of March 2024\nReviewer #2 (Remarks to the Author):\nI thank the authors for their considered responses and careful revision of the MS. I have no\nfurther comments and look forward to seeing the final published version soon."}]

Decision letter