Page 1

This is the Accepted Version of a paper published in the

journal Nature:

Reisz, Robert R., Huang, Timothy D., Roberts, Eric M.,

Peng, ShinRung, Sullivan, Corwin, Stein, Koen, LeBlanc,

Aaron R.H., Shieh, DarBin, Chang, RongSeng, Chiang,

ChengCheng, Yang, Chuanwei, and Zhong, Shiming (2013)

Embryology of Early Jurassic dinosaur from China with

evidence of preserved organic remains. Nature, 496. pp.

210-214.

http://dx.doi.org/10.1038/nature11978

ResearchOnline@JCU

Dinosaur embryology: inside the bones of an Early Jurassic

sauropodomorph from China

Robert R. Reisz

1*

, Timothy D. Huang

2

, Eric Roberts

3

, ShinRung Peng

4

, Corwin

Sullivan

5

, Koen Stein

6

, Aaron LeBlanc

1

, DarBin Shieh

4

, RongSeng Chang

7

, ChengCheng

Chiang

8

, ChuanWei Yang

9

, ShiMing Zong

10

1

Department of Biology, University of Toronto Mississauga, Mississauga, ON

L5L 1C6, Ontario, Canada.

2

National Chung Hsing University, Taichung 402, Taiwan.

3

School of Earth and Environmental Sciences, James Cook University, Townsville,

Queensland, QLD 4811, Australia.

4

Medical College Institute of Oral Medicine, National

Cheng Kung University, Tainan 701, Taiwan.

5

Key Laboratory of Evolutionary

Systematics of Vertebrates, Institute of Vertebrate Paleontology and Paleoanthropology,

100044 Beijing, China.

6

Steinmann Institüt für Geologie, Mineralogie und Paläontologie,

University of Bonn, 53115 Bonn, Germany.

7

Dept of Optics and Photonics, National

Central University, Chung-Li, 32001, Taiwan.

8

National Synchrotron Radiation Research

Center, Hsinchu 30076, Taiwan.

9

Lufeng County Dinosaur Museum, Lufeng, Yunnan,

China.

10

ChuXiong Prefecture Museum, 675000 Chuxiong, Yunnan, China.

Fossil dinosaur embryos are surprisingly rare, almost entirely restricted to Upper

Cretaceous strata that record the late stages of non-avian dinosaur evolution

1,2

.

Notable exceptions are the oldest known embryos from the Early Jurassic South

African sauropodomorph Massospondylus

3-4

, and Late Jurassic embryos of a

theropod from Portugal

5

. The fact that dinosaur embryos are rare and typically

enclosed in eggshells limits their availability for tissue and cellular level

investigations of development and growth. Consequently, little is known about

growth patterns in dinosaur embryos, even though post-hatching ontogeny has been

studied in several taxa

6

. Here we report the discovery of an embryonic dinosaur

bonebed from the Lower Jurassic of China, the oldest such occurrence in the fossil

record. The embryos are similar in geological age to those of Massospondylus and

are also assignable to a sauropodomorph dinosaur, probably Lufengosaurus

7

. The

unusual preservation of numerous disarticulated skeletal elements and eggshell in

this monotaxic bone bed, representing different stages of incubation and therefore

derived from different nests, provides opportunities for novel investigations of

dinosaur embryology in a clade noted for gigantism. For example, comparisons

among embryonic femora of different sizes and different developmental stages

reveal a consistently rapid rate of growth throughout development, possibly

indicating that short incubation times were characteristic of sauropodomorphs. In

addition, asymmetric radial growth of the femoral shaft and rapid expansion of the

fourth trochanter suggest that embryonic muscle activation played an important

role in the pre-hatching ontogeny of these dinosaurs. This discovery also provides

the oldest evidence of in situ preservation of complex organic remains in a

terrestrial vertebrate.

Monotaxic bonebeds are particularly prized by palaeobiologists because they yield

large numbers of bones that can reveal patterns of development and growth within a

single species

8

. Here we report the discovery of a monotaxic embryonic dinosaur

bonebed, from Lower Jurassic strata near Dawa, Lufeng County, Yunnan Province,

People’s Republic of China (specimens housed in the Chuxiong Prefectural Museum,

Catalogue No. C2019 2A233). These remains are equivalent in age to the oldest known

dinosaurian embryos, which belong to the South African sauropodomorph

Massospondylus

3

. However, the new Chinese sauropodomorph specimens differ in

comprising an accumulation of disarticulated skeletal elements representing various

stages of embryonic development, rather than a set of articulated skeletons enclosed in

eggs

9

. Thus, the earliest known dinosaurian embryo occurrences from China and South

Africa are mutually complementary, permitting different types of investigation into the

early stages of development in sauropodomorph dinosaurs that lived shortly after the end-

Triassic extinction.

The embryonic bonebed was discovered in the Dark Red Beds or Zhangjia’ao

Member

10, 11

of the Early Jurassic (Sinemurian, 190-197 Ma) Lower Lufeng Formation,

roughly 3-5 m below the top of the formation (Fig. 1). The Lower Lufeng Formation is

temporally, environmentally and faunally comparable to the Upper Elliot Formation of

southern Africa and the Kayenta Formation of North America

12

(Supplementary

Information 1). Like these similar rock units on other continents, the Lower Lufeng

Formation preserves abundant skeletal remains of basal sauropodomorphs

13

, and these

dinosaurs are commonly found in the upper part of the Dark Red Beds

14

.

Taphonomically, the 10-20 cm thick embryonic bonebed is characterized by the

presence of completely disarticulated skeletal elements at various stages of embryonic

development (Fig. 2), with calcium carbonate nodules often surrounding tightly packed

appendicular skeletal elements. One nodule contains a high concentration of eggshell

fragments that were apparently derived from soil compaction of a single egg. The latter

material provides for the first time microstructural information about the oldest known

terrestrial vertebrate eggshell (Supplementary Information 2, Supplementary Figs. 2.1-

2.5).

We interpret the bonebed as a para-autochthonous assemblage, formed by low-

energy flooding and slow inundation of a colonial nesting site. The host sediment is a

heavily bioturbated, massive siltstone, throughout which are dispersed isolated skeletal

elements, eggshell fragments and the small, fossil rich nodules of calcium carbonate. As a

result, there are no preserved nest structures or uncrushed eggs. The lack of coarse-

grained sediment, coupled with the apparent sorting and concentration of the bones (Fig.

2c, f, g), is intriguing from a taphonomic perspective. The bonebed does not appear to be

an in situ nest or catastrophic death assemblage, but is also not a time-averaged fluvial

deposit containing bones that were transported from beyond the immediate vicinity. The

latter possibility is incompatible with the high preservational quality of the delicate,

poorly ossified embryonic bones and <100 µm-thick eggshell, which would be expected

to suffer extensive damage during transport over substantial distances. However, the

presence of bones at different levels of development (i.e., from multiple clutches),

coupled with the concentration and partial alignment of eggshell fragments and certain

skeletal elements within the nodules (Fig. 2f), does indicate a degree of transport. We

believe that inundation, ponding, and partial decomposition, followed by weak currents

and simple wave action, represents the best explanation for the hydrodynamic sorting and

non-random orientation of the mostly disarticulated embryonic elements. The embryonic

bones and egg shells were eventually buried and subjected to pedogenic processes,

including bioturbation, soil compaction and expansion, and precipitation of carbonate

nodules around many of the bones.

The skeletal remains, more than 200 bones, include dozens of isolated cervical,

dorsal, and caudal centra, rib fragments, femora and other limb elements, scapulae, an

ilium, and a few skull elements (Fig. 2). These specimens are less ontogenetically

advanced in multiple respects than some previously known sauropodomorph, theropod

and ornithischian skeletons that can be definitively identified as embryonic because they

were discovered inside intact eggs, demonstrating that these specimens are embryos

rather than hatchlings

15

. Conspicuously embryonic features include the presence of teeth

that do not protrude beyond the alveolar edges of the maxilla and dentary, centra with

large notochordal canals and deeply pitted articular surfaces, and the universal presence

of extensive primary vascular spaces that are open to the surface

16-18

(Supplementary

Information 3, Supplementary Fig. 3).

The embryonic bones were compared with previously known saurischian and

ornithischian embryos, and found to share detailed resemblances with other

sauropodomorph embryos but not with embryos of ornithischians or theropods

1-3

. In

particular, identification of the Lufeng specimens as sauropodomorphan was greatly

facilitated by their similarity to the well known, articulated Massospondylus embryos

19

.

Interpretation of the embryonic bones as representing a basal sauropodomorph is based

not only on numerous features that are synapomorphies at various levels within basal

Sauropodomorpha, but also on the results of a phylogenetic analysis using data from a

recent study

20

. This analysis places the specimens well within the sauropodomorph clade

but well outside Sauropoda, and supports their tentative referral to the well-known

Lufeng Formation sauropodormorph Lufengosaurus. Within Sauropodomorpha, the

maxilla and its dentition show specific morphological resemblances to Lufengosaurus.

This identification is also supported by the presence of a possible femoral autapomorphy

(strong medial curvature of the distal part of the 4

th

trochanter) in the 24 embryonic

femora and in the holotype of L. huenei

7

. However, two other basal sauropodomorphs

have also been recovered from the Lower Lufeng Formation of Yunnan

14

, making referral

of the embryonic specimens to Lufengosaurus inescapably tentative. (Supplementary

Information 4 and Supplementary Figs. 4.1, 4.2).

Histological study of the Lufeng embryonic specimens provides an unprecedented

window into the process of embryonic growth in a dinosaur, because these fossils

represent numerous individuals at various stages of embryonic development. For

example, three thin-sectioned dorsal vertebrae show different stages in the embryonic

development of the notochordal canal

17

(Fig. 3), a feature that is absent in posthatchlings.

Longitudinal sections of two vertebrae (Fig. 3a, b) show that the cranial and caudal ends

consist mostly of hypertrophied calcified cartilage. The mid-regions of the vertebrae

show an initial stage of highly cancellous bone deposition, with numerous primary

cavities (“vascular spaces”), indicative of very fast growth

21

, and there is no evidence of

any bone remodeling. A transverse section through the third vertebra (Fig. 3c) shows the

notochordal canal as a large tunnel through the middle of the centrum, and reveals

erosion cavities that indicate resorption of the cartilaginous precursor. Small patches of

calcified cartilage are still visible.

The sample includes 24 femora, including 14 right femora (MNI=14). The femora

range from 2.6 to 4.5 mm in mid-shaft diameter and 12 to 22 mm in length, representing

individuals from multiple nests

19

, and permitting the first morphometric analysis of

embryonic growth and development in a dinosaur (Fig. 4a, Supplementary Information 5,

Supplementary Figs 5.1-5.4 ). Separate thin sections through the mid-shaft regions and 4

th

trochanters of three femora of different diameters also illustrate the development and

ossification of the femur (Fig.4a-c). The cross sections show major differences in

periosteal bone distribution, orientation of the vascular spaces (primary cavities), and size

and level of ossification of the 4

th

trochanter (insertion site of the primary propulsive

muscle of hindlimb). For example, in the smallest, least ossified femur (Fig. 4b), the 4

th

trochanter is small, in the mid-sized femur (Fig. 4c) the trochanter is more prominent, but

this femur shows little woven bone tissue in the trochanter, while in the largest femur

(Fig. 4d), the 4

th

trochanter is large and fully ossified. These successive stages of

embryological development have not been previously documented in dinosaurian

embryos. Mid-shaft cross sections of seven femora, including the three listed above (Fig.

4b-d) show a significantly greater degree of vascularity (ratio of primary cavity area to

total cross-sectional area of the cortex ranging from 56% to 65%) than in other dinosaurs,

indicating a sustained very rapid rate of growth

21

. In addition, the femoral medullary

cavity increases in diameter throughout embryonic development (Supplementary Fig.

5.4), indicating that, although the embryonic femur is composed entirely of primary bone,

reshaping by endosteal bone resorption in the medullary cavity occurs even at this early

stage of ontogeny. The high level of vascularity is the first known evidence that

sauropodomorph embryos probably grew at a faster rate than both extant birds and other

dinosaurs, a circumstance that may imply that sauropodomorphs had shorter incubation

times than their contemporaries. If this high rate of growth was maintained after hatching,

it might explain the ability of sauropodomorphs to consistently achieve larger adult size

than their dinosaurian contemporaries, and in some taxa reach gigantic proportions.

Extant vertebrates can display considerable limb and body movement prior to birth

or hatching, involving muscle activation that mediates skeletal development

22, 23

. In mice

and chickens this epigenetic phenomenon results in differential (asymmetrical) thickening

of the walls of the long bones for improved load-bearing, resulting in a condition similar

to that in the embryonic sauropodomorph femora. Similarly, sustained growth of skeletal

crests and flanges depends on activation of the muscles attached to them, an observation

that should be applicable to the dinosaurian 4

th

trochanter. It is likely that the uneven

thickening of the femoral walls in the Lufeng specimens, the circumferential orientation

of the primary vascular cavities, and the growth of the 4

th

trochanter (Fig. 4b-d) all

depended upon muscle contraction and embryonic motility, an important mechanism for

building a skeleton capable of coping with the functional demands encountered by the

neonate. This discovery adds the first fossil evidence of such epigenetic phenomena to a

growing body of research that documents their significance in extant model organisms.

The available isolated embryonic bones were also subjected to detailed analysis

using Synchrotron Radiation Fourier Transformation Infrared (SR-FTIR) spectroscopy

25

.

In contrast to previous studies that reported organic residues based on extracts obtained

by decalcifying samples of bone, our analyses (Fig. 5) were able to target particular

tissues in situ. Our approach made it possible to detect the preservation of organic

residues, likely direct products of the decay of complex proteins, within both the fast

growing embryonic bone tissue and the margins of the vascular spaces (Fig. 5a, b). This

is indicated by the multiple amide peaks revealed by both infrared (1,500-1,700 cm

-1

strong band from amide I & II, and 1,200-1,300 cm

-1

weak band from amide III) and

Raman spectroscopy (Amide A peak at 3,264 cm

-1

) (Supplementary Figs 6.1, 6.2).

Previous reports of dinosaur organic remains, or “dinosaurian soft tissues”

26-28

, have been

controversial because it was difficult to rule out bacterial biofilms or some other form of

contamination as a possible source of the organics

29, 30

. Our results clearly indicate the

presence of both apatite and amide peaks within the woven embryonic bone tissue (Fig.

5a), which should not be susceptible to microbial contamination or other postmortem

artefacts. Future work on the embryonic specimens will include more detailed analysis of

the nature of the organic remains that we have detected in these bones. However, their

preservation in such delicate, porous structures raises the very real possibility that other

fossils may also contain native soft tissues that can be studied with this targeted approach,

opening up new areas of palaeobiological research.

Methods Summary

Fossil preparation was done manually under a dissecting microscope. Thin sections

ranging from 30 to 50 µm in thickness were produced with minimal loss of tissue, and

photographed using a Nikon AZ100 microscope with a lambda filter. ImageJ software

was used to calculate the percentage vascularity of each femoral thin section, defined as

the ratio between the total area of the primary cavities and the overall area of the cortex

of the femur. NIS Elements imaging software for the Nikon AZ100 mounted digital

camera was used both for the photography and to confirm the percentage vascularity

calculations for a total of 8 femora. For FTIR analysis, infrared spectral line scans and

mapping data were collected using the SR-FTIR spectromicroscopy facility at the

National Synchrotron Radiation Research Center (NSRRC) beamline 14A1 (BL14A1) in

Taiwan. The spectra were recorded in reflectance mode from each sample section, using a

Thermo Nicolet Magna-IR spectrometer with the following settings: resolution 4 cm

−1

,

step-size 15 μm, aperture size 30 μm, and 128 scans. Peak position and baseline

corrections were performed using OMNIC peak resolving software after the results had

been obtained.

1. Carpenter, K., Hirsch, K., & Horner J. R. eds. Dinosaur Eggs and Babies. 372 pp.

(Cambridge Univ. Press, Cambridge, 1994).

2. Chiappe, L. M. et al. Sauropod dinosaur embryos from the Late Cretaceous of

Patagonia. Nature 396, 258-261 (1998).

3. Reisz, R. R., Scott, D., Sues, H.-D., Evans, D. C. & Raath, M. A.. Embryos of an

Early Jurassic prosauropod dinosaur and their evolutionary significance. Science 309,

761-764 (2005).

4. Reisz, R. R., Evans, D. C., Roberts, E. M., Sues, H.-D., & Yates, A. M. Oldest known

dinosaurian nesting site and reproductive biology of the Early Jurassic

sauropodomorph Massospondylus. Proc.Natl.Acad.Sci. USA 109, 2426-2433 (2012).

5. Ricqlès, A. D., Mateus, O., Antunes, M. T. & Taquet, P. 2001. Histomorphogenesis of

embryos of Upper Jurassic theropods from Lourinhã (Portugal). C. R. l´Acad. Sci.,

Paris, Earth and Planetary Sci. 332, 647-656 (2001).

6. Padian, K., de Ricqles, A. J. & Horner, J. R. Dinosaurian growth rates and bird

origins. Nature, 412, 405-408 (2001).

7. Young, C. C. A complete osteology of Lufengosaurus huenei Young (gen. et sp. nov)

from Lufeng, Yunnan, China. Palaeontol. Sinica, N S C 7:1–53 (1941).

8. Rogers, R. R., Eberth, D. A. & Fiorillo, A. R. eds. Bonebeds, Genesis, Analysis, and

Paleobiological Significance. Chicago Univ. Press, Chicago, 2007)

9. Carpenter, K. Eggs, nests, and baby dinosaurs. A look at dinosaur reproduction.

(Indiana Univ. Press, Bloomington, 1999).

10. Bien, M. N. “Red Beds” of Yunnan. Bull. Geol. Soc. China 21, 159–198 (1941).

11. Fang, X. et a. in Proceedings of the Third National Stratigraphical Congress of

China. 208–214 (Geological Publishing House, Beijing, 2000).

12. Weishampel, D. B., Dodson, P., and Osmólska, H. (eds.). 2004. The Dinosauria.

(Univ. California Press, Berkeley, 2004).

13. Sun, A. G. & Cui, K. H. in The Beginning of the Age of Dinosaurs. (ed. Padian, K. )

275-278 (Cambridge Univ. Press, New York, 1986).

14. Galton, P. M. & Upchurch, P. in The Dinosauria (eds. Weishampel D. B., Dodson,

P. & Osmolka, H.) 232-258 (Univ. California Press, Berkeley, 2004).

15. Kundrát, M., Cruickshank, A.R.I., Manning, T.W. & Nudds, J. Embryos of

therizinosauroid theropods from the Upper Cretaceous of China: diagnosis and

analysis of ossification patterns. Acta Zoologica (Stockholm) 89: 231–251. (2008).

16. Goodrich, E. S. Studies on the Structure and Development of Vertebrates. (Univ.

Chicago Press, Chicago, 1930).

17. Fleming, A., Keynes, R. J., &Tannahill, D. The role of the notochord in vertebral

column formation. J. Anat. 199, 177–180 (2001).

18. Francillon-Vieillot, H.,et al. in Skeletal biomineralization: Patterns, processes and

evolutionary trends. (ed. Carter, J. G) 471-530 (Van Nostrand Reinhold, New York,

1990).

19. Reisz, R. R., Sues, HD., Evans, D. C. & Scott, D. Embryonic skeletal anatomy of the

Early Jurassic prosauropod dinosaur Massospondylus. J. Vertebr. Paleontol. 30,

1653-1665 (2010).

20. Apaldetti, C., Pol, D., &Yates A. The postcranial anatomy of Coloradisaurus brevis

(Dinosauria: Suropodomorpha) from the Late Triassic of Argentina and its

phylogenetic implications. Palaeontology DOI: 10.1111/j.1475-4983.2012.01109.x

(2012).

21. Horner, J. R., Padian, K. & Ricqlès, A. D. Comparative osteohistology of some

embryonic and neonatal archosaurs: implications for variable life histories among

dinosaurs. Paleobiology 27, 39-58. (2001).

22. Müller, G. B. Embryonic motility: environmental influences and evolutionary

innovation. Evolution & Development 5, 56-60 (2003).

23. Sharir, A., Stern, T., Rot, C., Shahar, R. & Zelzer, E. Muscle force regulates bone

shaping for optimal load-bearing capacity during embryogenesis. Development 138,

3247-3259 (2011).

24. Blitz, E. et al. Bone ridge patterning during musculoskeletal assembly is mediated

through SCX regulation of Bmp4 at the tendon-skeleton junction. Developmental

Cell 17: 861-873 (2009).

25. Kong J. & Shaoning, Y. Fourier Transform Infrared Spectroscopic Analysis of

Protein Secondary Structures, Acta Biochimica et Biophysica Sinica 39(8), 549-

559 (2007).

26. Schweitzer, M. H., Wittmeyer, J. L., Horner, R. H. & Toporski J. K. Soft-Tissue

Vessels and Cellular Preservation in Tyrannosaurus rex. Science 307,1952-1955

(2005).

27. Schweitzer, M. H. et al. Analyses of soft tissue from Tyrannosaurus rex suggest the

presence of protein. Science 316, 277-280 (2007).

28. Lindgren J. et al. Microspectroscopic Evidence of Cretaceous Bone Proteins. PLoS

One 6, e19445 (2011).

29. Peterson, J. E., Lenczewski, M. E. & Scherer R. P. Influence of Microbial Biofilms

on the Preservation of Primary Soft Tissue in Fossil and Extant Archosaurs. PLoS

One 5, e13334 (2010).

30. Kaye, T. G., Gaugler, G. & Sawlowicz, Z. Dinosaurian soft tissue interpreted as

bacterial biofilms. PloS One 3(7), e2808 (2008).

Supplementary Information is linked to the online version of the paper at

www.nature.com/nature.

Acknowledgements We thank G. Grellet-Tinner, J. Steigler, P. Barrett, and E. Prondvai

for discussion, C. Chu and X.J. Lin for research support, S.P. Modesto and C. Brown for

field assistance, D. Scott for specimen preparation and photography, N. Campione for

morphometric analysis, C. Apaldetti for data matrix, J.R. Liu for assistance in Lufeng,

and C.C. Wang, Y.F. Song, Y.C. Lee, and H.S. Sheu for help with various experiments at

the National Synchrotron Radiation Research Center, Taiwan. Research support was

provided by DFG FOR 533 (Germany), NSC 100-2116-M-008-016 (Taiwan), Ministry of

Education (Taiwan) under the NCKU Aim for the Top University Project, NSERC

Discovery and SRO Grants (Canada), University of Toronto, Chinese Academy of

Sciences and National Natural Science Foundation of China.

Author Contributions R.R.R jointly conceived and designed the project with T.D.H.;

R.R.R. wrote paper, supervised preparation and scientific illustration of specimens;

T.D.H., E.R., C.S., K.S., A.L., contributed to manuscript; R.R.R., T.D.H., E.R., C.S.,

R.S.C, and C.W.Y. contributed significantly to field work; T.D.H., S.R.P., D.B.S.

supervised and completed multimodal optical and chemical spectroscopic analyses; K.S.,

A.L., C.C.C. prepared slides and illustrated thin sections; R.R.R., T.D.H., K.S., E.R.,

S.R.P., C.S. wrote Supplementary Information; T.D.H., R.S.C., C.W.Y., S.M.Z. provided

logistical support for field work and research.

FIGURE CAPTIONS:

Figure 1. Location and stratigraphy of Lufeng monotaxic embryonic bone bed. a,

Map of China, with study area in Yunnan Province shown by inset box. b, General

geological map of study area. c, Stratigraphic section showing location of embryonic

bone bed within Zhangjia’ao Member of Lower Lufeng Formation.

Figure 2. Sauropodomorph dinosaur embryonic skeletal elements derived from the

Lufeng bone bed. a, Reconstructed embryonic skeleton of Early Jurassic

sauropodomorph (using Massospondylus as a model, not to scale), showing in dark red

the elements known from Dawa (Chuxiong Prefectural Museum, Specimen No. C2019

2A233). Numerous ribs, centra, and distal limb elements are known, but their exact

locations within the skeleton are difficult to determine. b, Left maxillae in ventromedial

and labial views, respectively, with enlarged view of partially erupted tooth. c, mid-dorsal

centrum in lateral and anterior views. d, Left ilium in lateral view. e, Right scapula,

vertebrae, and left humerus preserved in one nodule. f, Right femur in posterolateral and

medial views, showing prominence and shape of 4

th

trochanter. g, Large right femur

preserved with ribs and various other skeletal elements in nodule. h, Embryonic limb

elements and ribs showing alignment along long axes. i, Close-up of proximal end of

right tibia, showing external foramina of primary cavities (also called vascular canals).

Scale bar = 1cm, unless otherwise shown.

Figure 3. Embryonic vertebral histology (C2019 2A233). a, Largest dorsal centrum,

longitudinal section, cranial portion showing initial closure of notochordal canal (cd), and

presence of erosion cavities (ee) with endochondral bone in the calcified cartilage (cc). A

collar of periosteal bone (pb) has already been formed. b, Smallest dorsal centrum,

longitudinal section showing whole length of bone, and representing earlier embryonic

stage with widely open notochordal canal. c, Intermediate-sized dorsal centrum,

transverse section, showing notochordal canal in cross section within substance of

vertebral centrum (vc). Neural canal (nc) visible directly above notochordal canal. All

scale bars equal 500 µm. Photographs taken with cross polar light with lambda filter.

Figure 4. Embryonic femoral morphometric analysis and histology (C2019 2A233).

a, Results of regression analysis showing growth trajectory of femoral mid-shaft diameter

relative to length of femur on left, and box-plot of residuals on right. Black dots represent

Lufeng bonebed embryonic femora, blue dots represent two embryonic and one hatchling

femora of the basal sauropodomorph Massospondylus, and red dot represents a hatchling

femur of the sauropodomorph Mussaurus. The regression analysis is based solely on the

Lufeng bonebed femora and shows a strong correlation between femoral length and shaft

diameter, and there are no outliers in this sample. However, all specimens of the other

two sauropodomorphs are well outside the range of variation exhibited by the Lufeng

material, and exhibit thinner femora relative to their respective lengths. This is likely

related to their presumed smaller egg, and eventual smaller and more gracile adult size

(see Supplemental Information 5 and Supplemental. Figs 5.1-5.4 for data and additional

morphometric analyses). b-d, Smallest to largest femora, sectioned transversely at mid-

diaphysis and level of 4

th

trochanter, from left to right. Photographs taken with cross

polar light with lambda filter. Abbreviations: m, medullary cavity; tr, 4

th

trochanter; nf,

nutrient foramen; pb, primary periosteal bone with primary cavities (“vascular canals”).

Figure 5. Synchrotron Radiation Fourier Transformed Infrared Spectroscopic (SR-

FTIR) analysis of embryonic femur, targeting different points within a small area of

the bone (a-c). Images in each row include, from left to right: mosaic composed of 12

individual optical IR images (150x180 µm each), showing total FTIR scanned area (red

box), specific point targeted for analysis (red cross) and 15 µm step size (red dots); 2D

and 3D FTIR distributions of absorption for the spectral band showing the highest

intensity at the targeted point, with blue and red corresponding to low and high

absorption, respectively; and K-K correction processed IR spectrum for targeted point. a,

FTIR scan targeting primary bone tissue, showing an apatite peak within the primary

bone but not within the vascular spaces; primary bone tissue also shows an amide peak at

1,500-1,700 cm

-1

, within the apatite crystal. b, FTIR scan targeting edge of vascular

space, near primary bone tissue, showing that the margins of the vascular spaces are

characterized by a 1,537 cm

-1

amide I & II peak but lack an apatite peak. c, FTIR scan

targeting central area of a vascular canal, showing an 885 cm

-1

carbonate peak within the

vascular spaces; a modest amide peak at 1,537 cm

-1

is also present, and we interpret the

carbonate peak as the result of calcite infilling of the vascular canals.