Report

Specialized Craniofacial Anatomy of a Titanosaurian

Embryo from Argentina
Graphical Abstract Authors
Martin Kundrát, Rodolfo A. Coria,
Terry W. Manning, Daniel Snitting,
Luis M. Chiappe, John Nudds,
Per E. Ahlberg

Correspondence
[email protected]

In Brief
Kundrát et al. report the first 3D preserved
embryo representing a sauropod
sauropodomorph. The analyses indicate
an unusual monocerotic face and the
early development of stereoscopic vision
for a sauropod. It reveals significant
heterochrony in prenatal cranial
ossification when compared with non-
sauropod sauropodomorphs.

Highlights
d The first 3D preserved embryonic skull of a sauropod
sauropodomorph

d It differs in facial anatomy and size from the sauropod

embryos of Auca Mahuevo

d It exhibits an unusual monocerotic and stereoscopic face for

a sauropod

d Heterochronies in cranial ossifications occurred in the

evolution of sauropods

Kundrát et al., 2020, Current Biology 30, 1–7

November 2, 2020 ª 2020 Elsevier Inc.
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Specialized Craniofacial Anatomy
of a Titanosaurian Embryo from Argentina
Martin Kundrát,1,2,7,* Rodolfo A. Coria,3 Terry W. Manning,4 Daniel Snitting,2 Luis M. Chiappe,5 John Nudds,6
and Per E. Ahlberg2
1Evolutionary Biodiversity Research Group, PaleoBioImaging Lab, Center for Interdisciplinary Biosciences, Technology and Innovation Park,


Pavol Jozef Safárik University, Jesenná 5, Ko
sice 04154, Slovak Republic
2Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Norbyva €gen 18A, Uppsala 75236, Sweden
3CONICET – UNRN - Museo Municipal ‘‘Carmen Funes,’’ Av, Córdoba, Plaza Huincul 8318, Neuque n, Argentina
4Freelance Paleo Technician, PO Box 697, Cortaro, AZ 85652, USA
5Natural History Museum of Los Angeles County, 900 Exposition Boulevard, Los Angeles, CA 90007, USA
6Department of Earth and Environmental Sciences, The University of Manchester, Oxford Road, Manchester M13 9PL, UK
7Lead Contact

*Correspondence: [email protected]
https://doi.org/10.1016/j.cub.2020.07.091

SUMMARY

The first dinosaur embryos found inside megaloolithid eggs from Auca Mahuevo, Patagonia, were assigned
to sauropod dinosaurs that lived approximately 80 million years ago. Discovered some 25 years ago, these
considerably flattened specimens still remain the only unquestionable embryonic remains of a sauropod
dinosaur providing an initial glimpse into titanosaurian in ovo ontogeny. Here we describe an almost intact
embryonic skull, which indicates the early development of stereoscopic vision, and an unusual monocerotic
face for a sauropod. The new fossil also reveals a neurovascular sensory system in the premaxilla and a partly
calcified braincase, which potentially refines estimates of its prenatal stage. The embryo was found in an egg
with thicker eggshell and a partly different geochemical signature than those from the egg-bearing layers
described in Auca Mahuevo. The cranial bones are comparably ossified as in previously described speci-
mens but differ in facial anatomy and size. The new specimen reveals significant heterochrony in cranial os-
sifications when compared with non-sauropod sauropodomorph embryos, and demonstrates that the
specialized craniofacial morphology preceded the postnatal transformation of the skull anatomy in adults
of related titanosaurians.

RESULTS AND DISCUSSION MCF-PVPH-874 represents a fragment of a fossil egg and the
egg-filling sediments with the skull in situ (Figures 1A, 1B, and
First 3D Preserved Sauropod Sauropodomorph Embryo S1). No postcranial remains were recognized inside the egg as
and Its Repatriation in other embryonic specimens from Auca Mahuevo (Figure 1C)
Sauropodomorph embryology remains one of the least explored [1]. The skull has been well exposed on its left side after chemical
areas of the life history of dinosaurs. The first definitive discovery preparation, but we have also imaged the skull with propagation
of sauropod embryos came with the finding of an enormous nest- phase contrast synchrotron microtomography (Figures 1D, 1E,
ing ground of titanosaurian dinosaurs known as Auca Mahuevo, S2, and S3; Video S1) at the European Synchrotron Radiation
discovered in Upper Cretaceous deposits of northern Patagonia, Facility (ESRF) in Grenoble, France. The scan revealed many
Argentina [1, 2]. Recently, embryonic remains of the early sauro- previously unknown anatomical details and the internal structure
podomorphs Massospondylus and probably Lufengosaurus were of the preserved bones and putative soft tissue (Figure S4).
reported from the Early Jurassic of South Africa [3] and China [4], Here we report on this exceptional specimen of sauropod em-
respectively. Our study contributes yet another important spec- bryo that exhibits cranial bones including braincase components
imen for understanding sauropodomorph ontogeny, in the form keeping their original shape, mostly intact surfaces and articula-
of a new specimen of titanosaurian embryo from Argentina, albeit tion. These circumstances enabled us (1) to reconstruct the most
of unknown provenance within Patagonia. The original egg was plausible appearance of the skull in titanosaurian sauropods
illegally exported from this country, and eventually brought to before hatching; (2) to describe anatomical characters based
the attention of one of us (T.W.M.). After preparation exposed em- on intact cranial bones useful for taxonomic or ontogenetic com-
bryonic remains, the unique preservation and scientific impor- parisons; (3) to revise opinions on how babies of these giants
tance of the specimen became evident and T.W.M. repatriated may be hatched, in particular, if this process was facilitated by
the fossil to the Museo Municipal ‘‘Carmen Funesˮ in Plaza Huin- the rostral horn [5]; (4) to test some previous suggestions about
cul, Neuque n Province, Argentina, where the specimen was cata- sauropodomorph reproduction [6] and cranial ossification [7];
logued under the number MCF-PVPH-874. and (5) to reveal biological and geochemical characters that

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Figure 1. The New Specimen of Megalooli-
thid Egg Containing the 3D Preserved Skull
of a Titanosaurian Embryo MCF-PVPH-874
(A) Sediment filling the egg with the embryonic
skull in situ.
(B) Magnified perspective of the embryonic skull
with the preorbital and orbital region in left lateral
view.
(C) The previously described flattened embryonic
skull MCF-PVPH-263 from Auca Mahuevo.
(D and E) Digital reconstruction of the cranial
bones: D, in lateral view, and E, in medial view (see
Video S1 and Figures S1–S3); notice mineralized
portions of neurocranial bones (see Figure S4).
(F) Reconstruction of the skull in anterior view
showing incomplete skull roof and antero-lateral
orientation of orbits; notice the preserved pre-
maxilla-maxilla suture, which suggests that the
narial opening is anteriorly limited by the maxilla,
with the premaxilla possibly restricted to the
sagittal contact.
(G) The left part of the skull in anterior view; notice
placement of the orbit and the posteriorly re-
tracted narial opening (ellipse). af, antorbital
fenestra; an, angular; bo, basioccipital; cp, cultri-
form process of the parabasisphenoid; d, dentary;
eo, exoccipital; ept, ectopterygoid; f, frontal; itf,
infratemporal fenestra; j, jugal; la, lacrimal; ls, lat-
erosphenoid; m, maxilla; mf, mandibular fenestra;
n, nasal; no, narial opening; orb, orbit; p, parietal;
paf, preantorbital foramen; pal, palatine; pe,
postdental emargination; pm, premaxilla; pmh,
premaxillary horn; po, postorbital; prf, prefrontal;
pro, prootic; pt, pterygoid; qj, quadratojugal; q,
quadrate; san, surangular; sp, splenial; sq, squa-
mosal; vo, vomer.

The calcified neurocranial elements

provide some morphological clues about
their identity and potentially refine esti-
mates of a prenatal stage of the titano-
saurian skull. The elements identified as
basioccipital, prootic, and exoccipital
are comparably as incomplete as those
distinguish the new specimen from previously described titano- that occur around days 47 to 50 of in ovo development of the alli-
saurian embryos from Auca Mahuevo [1, 2]. gator neurocranium (Figure S4E). At hatching, the neurocranium
is usually well ossified in the alligator, whereas small cartilagi-
Cranial Ossifications and Stage Assessment nous streaks remain between basicranial bones in precocial spe-
Most of the MCF-PVPH-874 skull is well ossified except for the cies of modern birds [9].
roof (Figures 1F and 1G). The skull roof fenestra remains largely Adopting the bone mineralization scale of the modern
open and the bone ossifications surrounding it are slightly in precocial archosaurs provides a simplified, but so far the
advance of the condition seen in a 50-day embryo of the Amer- only, framework for reasonable assessment of the develop-
ican alligator [8]. Notably, the retroarticular process (posterior mental age of dinosaur embryos [10]. Assuming the pres-
end of the Meckel’s cartilage) starts to mineralize around day ence of developmental precociality in titanosaurians
44 in the alligator [8], whereas no fossilized remains of the artic- (due to hypothetical lack of any parental care), embryo
ular have been observed in MCF-PVPH-874 (SOM). Further- MCF-PVPH-874 has already undergone four-fifths of its in
more, we found that most of the embryonic braincase elements ovo development. The absolute length of the incubation
of MCF-PVPH-874 were lightly calcified and preserved at the period, however, remains unknown for any sauropod
stage of acquiring their surface contours at the time of death. dinosaur.
The only elements with definable outlines include the cultriform Recently, embryos of the non-sauropod sauropodomorph
process of the parabasisphenoid and the laterosphenoid Massospondylus carinatus (BP/1/5347a) were found to be
(Figure S4D). only 60% through their incubation period (IP) using cranial

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Figure 2. Whole Rock Geochemistry of the
Eggshell and Sediment Samples from North-
ern Patagonia
(A) Comparisons of X-ray fluorescence-based an-
alyses of MCF-PVPH-874 and three samples
from Layer 3 of Auca Mahuevo; notice significantly
lower amount of several specific oxides in MCF-
PVPH-874.
(B) High-resolution X-ray diffractometry spectrum
of the Auca Mahuevo specimen showing the pres-
ence of the tectosilicate mineral analcime (orange
colored peaks and arrows).
(C) High-resolution X-ray diffractometry spectrum
of MCF-PVPH-874; notice the absence of the
analcime.

Geochemistry Signatures
To investigate the provenance of MCF-
PVPH-874 further, we analyzed the
whole-rock geochemistry of three sam-
ples of fossil eggshell and sediment from
Layer 3 of Auca Mahuevo and one sample
derived from MCF-PVPH-874. The X-ray
fluorescence (XRF) analysis revealed that
all samples from Auca Mahuevo contain
higher amounts of several specific oxides
than sample MCF-PVPH-874 (Figure 2A).
The samples were also analyzed by X-ray
diffractometry (XRD) to identify the prin-
cipal minerals present. The XRD spectra
revealed the presence in all Auca Mahuevo
samples of the tectosilicate mineral anal-
cime (Figure 2B).
Analcime is indicative of the lowest
(zeolite) grade of metamorphism or
diagenesis of a sedimentary facies. Such
diagenesis would happen on a regional
scale. The XRD spectra thus suggest that
there has been pervasive zeolitic alteration
at Auca Mahuevo. It is inconceivable that
the analcime is detrital, for example
washed into the basin from elsewhere, as
this mineral is exceedingly rare in source
rocks and is soft and easily broken down.
It is most probably a widespread diage-
netic product as a result of decomposi-
tion/alteration of the less stable silicates
[12]. Analcime, however, is totally absent
ossification sequences of living saurians [7]. Having compared from the sample of MCF-PVPH-874 (Figure 2C), indicating that
MCF-PVPH-874 (75% of IP) with BP/1/5347a (60% of IP), this specimen did not undergo such regional diagenesis/meta-
we suggest that significant developmental heterochronies be- morphism and derives from a different locality than the egg layer
tween snout and calva had occurred in the evolution of titano- of Auca Mahuevo 3 [2].
saurian sauropods. In fact, MCF-PVPH-874 exhibits rather a
reversed ossification pattern of advanced ossifications of the Eggshell Microstructure and Classification
snout (contra incomplete sutures in BP/1/5347a). Moreover, Embryo MCF-PVPH-874 was found inside an egg referable to
MCF-PVPH-874 also shows much delayed ossification of Megaloolothidae [13] (Figures 3A and 3B). The monolayered
posterior roofing bones as in crocodilians [8] and birds eggshell consists of fan-shaped units, whose tops constitute
[11], contra complete interfrontal and interparietal sutures in tubercular surface ornamentation (Figure 3B). Having compared
BP/1/5347a. the eggshell parameters among megaloolithid eggs from several

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Figure 3. Eggshell Microstructure of MCF-
PVPH-874
(A) The fragment of the original egg.
(B) Radial thin section through the monolayered
eggshell of the megaloolithid type showing fan-
shaped units.
(C) Inner surface of the eggshell exposing the
coalescent mammillary tips with central concav-
ities (arrow).
(D) The coalescent inner (mammillary) tips of the
eggshell units in radial view; notice a fibrous mat
underlying the innertips.
(E) The same perspective in polarized light
revealing deep resorption pits (asterisk); notice a
close association between the innertips and un-
derlying fibrous structure that likely represents the
shell membrane (arrow).

sites in northern Patagonia, we found that MCF-PVPH-874 has a premaxilla-maxilla suture inclines postero-dorsally, contrasting
significantly thicker eggshell (1.6–2 mm) than the specimens with a short snout and vertically oriented suture in MCF-PVPH-
from Auca Mahuevo (0.6–1.3 mm) [6]. The eggshell thickness is 272 and 263. A preantorbital fenestra of MCF-PVPH-874 opens
more similar to specimens from the Mansilla I and II localities ventral rather than posterior to the rostral rim of the antorbital
(1.8–2 mm) in the Rı́o Negro province [14]. It is also comparable fenestra (character 2); this condition is also seen in Tapuiasaurus
to the partial egg specimen (1.7–2.1 mm) found near Neuque n [16]. The lacrimal of MCF-PVPH-874 projects more vertically
City (the same stratigraphic unit that contains Auca Mahuevo) (character 3) as in skeletally mature specimens (see below),
[15]. and contrasts markedly with the rostrocaudally inclined lacrimal
Finally, we observed significant central concavities on inner in MCF-PVPH-272 and 263. Furthermore, a rostrodorsal rim on
tips of single and coalescing shell units in MCF-PVPH-874 (Fig- the postdental emargination is slightly angled (character 4) in
ure 1C) contrasting with an absence of any sign of calcium MCF-PVPH-874, as in Tapuiasaurus [16], Antarctosaurus [17],
resorption in specimen MCF-PVPH-263 from Auca Mahuevo. Sarmientosaurus [19], and Rapetosaurus [20], whereas it is
The resorption pits are deeply excavated and merged in a fibrous notched in the embryos MCF-PVPH-272 and MCF-PVPH-263
mat that probably represents the fossilized remains of the shell and in Bonitasaura [17]. The jugal of MCF-PVPH-874 has a
membrane (Figures 3D and 3E). Thus, we provide for the first deeply incised caudal margin (character 5) and recalls the condi-
time evidence that titanosaurian embryos utilized eggshell- tion in Tapuiasaurus [16]. Unlike MCF-PVPH-272 and 263 [2, 21],
derived calcium relatively long before they approached the the quadratojugal and squamosal contact directly (character 6)
hatching stage. as in titanosaurians from South America and Africa [16–20].
These characters most probably had undergone post-hatching
Developmental Disparity in the Evolution of remodeling to some extent, and should therefore be used with
Titanosaurian Face caution when searching for relatives of MCF-PVPH-874 among
Embryo MCF-PVPH-874 shows one of the most complete and known titanosaurian taxa. It appears that embryo MCF-PVPH-
articulated skulls known from a titanosaurian dinosaur from 874 is most similar in morphology to Tapuiasaurus (characters
Patagonia. In fact, only a few titanosaurian skulls are known 1, 2, and 4–6) from the Early Cretaceous of Brazil [16], suggesting
globally, including that of Tapuiasaurus macedoi [16] from Brazil; a hypothetical affinity of MCF-PVPH-874 to nemegtosaurid
Bonitasaurus salgadoi [17], Antarctosaurus wichmannianus [18], titanosaurians.
and Sarmientosaurus musacchioi [19] from Patagonia; and Differences between the new titanosaurian embryo and previ-
Rapetosaurus krausei from Madagascar [20]. MCF-PVPH-874 ously described specimens [1, 2, 17] are also expressed in quan-
is cranially most similar to the previously described embryos titative terms. In terms of size proportions, MCF-PVPH-874 is
from Auca Mahuevo, MCF-PVPH-272 and MCF-PVPH-263 significantly smaller (25%–35%) than MCF-PVPH-263. In com-
[1, 2]. It is assigned to titanosaurians based on the presence of parison to the latter, the new specimen has a shorter dorsoven-
several characters such as a distinct notch ventral to the antor- tral diameter of the orbit (8.9 mm versus 11.9 mm), antero-pos-
bital fenestra (postdental emargination), a rostrally extended terior length of the antorbital fenestra (4.1 mm versus 6.4 mm),
quadratojugal, and the presence of a mandibular fenestra and the maximum length of the postorbital (8.4 mm versus
[1, 2, 21]. 10.9 mm; measured from the anterior contact with the jugal to
Noteworthy are the number of anatomical details that differ in the most postero-dorsal extension of the postorbital). Despite
MCF-PVPH-874 from the two previously described specimens the metric differences, the cranial ossification patterns are
(SOM). The maxilla is rostrally longer (character 1) and the almost identical. A reasonable explanation of this developmental

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Figure 4. Reconstruction of the Craniofa-
cial Anatomy of the MCF-PVPH-874 Titano-
saurian Embryo
(A) Placement of the egg tooth in modern bird
embryo.
(B) Reconstruction of the head appearance by
Vladimı́r Rimbala; the blue arrow points to the
probable placement of the keratinous egg-tooth
and the red arrow to the premaxillary horn
including both keratinous and bone components.
(C) The craniofacial region in ventral view showing
the premaxillary and maxillary alveoli and the
rostral premaxillary projection forming a basis of
the horn-like process (red arrow).
(D) The skull in antero-ventral view; notice rostral
pointed projection (red arrow).
(E) 3D rendered first mesial premaxillary teeth.
(F and G) 3D rendering of the opaque and semi-
transparent premaxilla in medial and lateral views;
notice distribution of the neurovascular canals,
premaxillary teeth in situ, and the premaxillary
horn (red arrow). See also Figure S1.

limit of the dentary recalling growth

disparity of the upper and lower jaw in
modern birds (Figures 4A and 4B). Unlike
modern birds, the rostral process pro-
jects dorsal to the rostral rim of the pre-
maxilla including alveoli and preserved
teeth in MCF-PVPH-874 (Figures 4C–4E
and S1D). Therefore, in contrast to previ-
ous reconstructions of MCF-PVPH-272
and 263 [5], we suggest that the rostral
process lacks occlusion with dentary
teeth and is actually free of alveoli. It is
disparity could be that the ossification rates of MCF-PVPH-874 likely that a thicker corneous sheet developed over the rostral
increase developmental variability in the same species that process and gave a horn-like appearance to the embryonic
could correspond to different incubation and environmental con- face of MCF-PVPH-874 (see red arrow in Figure 4B).
ditions. Alternatively, MCF-PVPH-874 could represent a titano- Fourth, unlike adults, the orbits face anterolaterally in the em-
saurian taxon different to that of MCF-PVPH-263 and 272. bryo MCF-PVPH-874 (Figures 1F and 1G). Thus, we assume that
early juveniles of titanosaurian sauropods might benefit from a
In Ovo Cranial Development temporary ability of at least a partial binocular vision that would
MCF-PVPH-874 further provides the first 3D perspective of a provide a much better visual perception. Putative stereoscopy
sauropod sauropodomorph embryo. The preserved configura- was suggested for Bajadasaurus, a sauropod from Patagonia
tion of cranial bones reveals several breakthroughs in our under- [22]; however, it was assumed based on a rather dorsal than
standing of the early development of the titanosaurian skull. anterior placement of orbits. The stereoscopy with ‘‘dorsally’’
First, it shows that some of the characters discussed above (1 projecting orbits became functional (e.g., in predator detection)
and 3) may be less different among the titanosaurian embryos if either when the head was held with the snout facing ventrally
structures are compared and viewed at corresponding angles. on an erected neck or when the animal was grazing and the
Thus, we refine the appearance of these characters based on eyes faced anteriorly.
the 3D preservation of MCF-PVPH-874 and interpret the differ-
ences with the Auca Mahuevo specimens as artifacts due to The Titanosaurian Premature Monocerotic Face
postmortem deformation. The interior of the premaxilla is canalized by a set of neurovascu-
Second, it reveals that several transforming morphologies lar canals radiating from the original ossification centrum with the
such as an elongated snout and retracted external narial open- two longest canals projecting toward the rostral process (Figures
ings, which have been hypothesized to appear during a juvenile 4F and 4G). Three neurovascular branches project toward the
period of titanosaurian ontogeny [5], are in fact already present in external surface; such configuration most probably supported
the MCF-PVPH-874 embryo prior to hatching. cell proliferation and sensory functions of the overlying skin.
Third, the premaxilla is slightly vaulted dorsally and forms a Another set of neurovascular canals projects lingually and termi-
massive broad-based spike protruding far beyond the rostral nates in the alveolar bone. Beside those, there is a single, the

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longest, neurovascular canal that extends to the base of the pre- STAR+METHODS
maxillary horn on the ventral side (Figure 4G).
The bony rostral process of the premaxilla has been inter- Detailed methods are provided in the online version of this paper
preted as a functional analog [5] of the keratinous egg-tooth and include the following:
seen in modern crocodiles and birds [23–25]. In contrast to
this, a functional egg tooth (consisting of dentin and enamel) de- d KEY RESOURCES TABLE
velops from rudiments laid down in the premaxillary bone and d RESOURCE AVAILABILITY
grows at significantly higher rates exceeding other teeth in size B Lead Contact

in Squamata [26]. Furthermore, two functional egg teeth were re- B Materials Availability
B Data and Code Availability
ported in Gekkota [26], increasing the developmental diversity of
the overall concept of egg teeth. d EXPERIMENTAL MODEL AND SUBJECT DETAILS
We do not know the exact in ovo position of the head of d METHOD DETAILS
B Provenance of the analyzed specimen
MCF-PVPH-874; however, given the curled position of extant
B Chemical Preparation
reptiles [27, 28] of comparable ossification stage and dorsally
projected egg tooth, it might be difficult to explain how the B Geochemistry
B Physical thin-sectioning
rostral process facilitated breaking the eggshell during hatching
B Scanning electron microscopy
in titanosaurians. Consequently, if an egg tooth was retained in
titanosaurian embryos, it seems more likely that it developed B Wholemount skeletal staining

on the dorsal surface of the snout, regardless of the presence B 3D Imaging

of a premaxillary horn-like structure. The most plausible topo-

graphic placement of a functional egg-tooth would be over SUPPLEMENTAL INFORMATION
the vaulted dorsal surface of the premaxilla (see blue arrow in
Supplemental Information can be found online at https://doi.org/10.1016/j.
Figure 4B). This egg tooth would likely have been shed shortly
cub.2020.07.091.
after hatching, whereas the bony premaxillary horn probably
persisted for some time in juvenile titanosaurians from
ACKNOWLEDGMENTS
Patagonia.
In summary, embryo MCF-PVPH-874 contains the best 3D in We thank the European Synchrotron Radiation Facility for providing instru-
ovo preserved embryonic skull of a sauropod sauropodomorph ments and funding; P. Tafforeau for his important role for scanning of the spec-
dinosaur or dinosaur in general. It is noteworthy that MCF- imen with assistance of M.K., data reconstruction, and processing; P. Sec
kar
PVPH-874 shows significant heterochronic changes in ossifica- for physical sectioning; J. Perran Ross for providing the embryos of Alligator
mississippiensis; V. Rimbala for the embryo reconstruction under supervision
tion of cranial bones when compared with the non-sauropod
of M.K.; P. Lythgoe and A. Bewsher for XRF analyses; J. Waters for XRD
sauropodomorph embryos [7]. It appears that the embryos of spectra; D. Green for mineralogical advice; and S. Abramowicsz for photo-
the massospondylid sauropods develop the skull roof before graphs used in Figures S1 and S4. The authors thank all reviewers for
the snout while the embryos of titanosaurian sauropods exhibit constructive critique and valuable comments that helped to improve this pub-
the reversed pattern. Given the fact that we do not know how lication. This work was funded by the Swedish Research Council via a Lin-
long these sauropod embryos developed inside their eggs, it is naeus Framework Grant ‘‘The Genomics of Phenotypic Diversity in Natural
Populations,ˮ awarded to P.E.A., and by the Slovak Research and Develop-
difficult to assess how much these prenatal heterochronies re-
ment Agency under the contract no. APVV-18-0251 and Scientific Grant
mained expressed in the cranial morphology at hatching. We as-
Agency VEGA of the Ministry of the Education, Science, Research and Sport
sume, however, that the delayed closure of the skull roof in the of the Slovak Republic (1/0853/17) awarded to M.K.
titanosaurian sauropods might be linked with development of
the dorsal dural sinus that is a prominent endoneurocranial AUTHOR CONTRIBUTIONS
structure in sauropods [29].
The specimen MCF-PVPH-874 implies that titanosaurian M.K. designed the research project; D.S. and M.K. prepared 3D models;
hatchlings hatched with a temporary monocerotid (single- T.W.M. prepared the specimen chemically; J.N. provided geochemical anal-
horned) face, posterodorsally retracted narial openings, and ysis data; M.K. wrote the manuscript with contributions by J.N., D.S.,
R.A.C., P.E.A., and L.M.C.
early binocular vision. Although we do not disregard the hatch-
ing scenario of titanosaurian embryos using the boney ‘‘egg-
DECLARATION OF INTERESTS
toothˮ sensu Garcı́a [5], our observations of MCF-PVPH-874
make it equally reasonable to consider other alternatives such The authors declare no competing interests.
as using the epidermal prominence (thickening located on the
median line between the narial and jaw edge) as in modern Received: April 8, 2020
crocodilians and birds or by taking advantage of temporary hy- Revised: June 16, 2020
pertrophied musculature. Furthermore, the new specimen Accepted: July 29, 2020
Published: August 27, 2020
shows the first real cranial geometry of a titanosaurian
sauropod from South America and reveals a putative affinity
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Current Biology 30, 1–7, November 2, 2020 7

 Please cite this article in press as: Kundrát et al., Specialized Craniofacial Anatomy of a Titanosaurian Embryo from Argentina, Current Biology (2020),
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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Fossil Samples
3D in ovo preserved titanosaurian embryo This paper MCF-PVPH-874
Flattened in ovo titanosaurian embryo [1, 2, 16] MCF-PVPH-263
Flattened in ovo titanosaurian embryo [1, 2, 16] MCF-PVPH-272
Biological Samples
Embryos of Alligator mississippiensis This paper + [7] CIB-Am1-4
Deposited Data
Scanned Data http://paleo.esrf.eu N/A
Software and Algorithms
Mimics v.12.3 and v.13.1 Materialise, Belgium N/A
VGStudio Max 2.2 Volume Graphics, Germany N/A
Blender v.2.78 https://www.blender.org/ N/A

RESOURCE AVAILABILITY

Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Martin
Kundrát ([email protected]).

Materials Availability
The MCF-PVPH-874 specimen is available for study at Museo Carmen Funes at Plaza Huincul in Argentina.

Data and Code Availability

The scanned data of the MCF-PVPH-874 specimen will be made publicly available on the ESRF paleontology online database at
http://paleo.esrf.eu. The published article includes all codes generated or analyzed during this study.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

The described specimen, MCF-PVPH-874, was analyzed from first-hand observations including 3D virtual high-resolution models.
The fossil has been repatriated back to Argentina and has become an integral part of the collection of Museo Municipal ‘‘Carmen
Funes’’ in Plaza Huincul where it will be available for future investigation.
The experimental subjects include the new 3D in ovo preserved specimen of titanosaurian embryo (MCF-PVPH-874) described in
this study and two previously described [1, 2] flattened in ovo preserved titanosaurian specimens (MCF-PVPH-263 and 272) for
comparative reasons. These specimens are housed at the Museo Municipal ‘‘Carmen Funes’’, Avenida Córdoba, Plaza Huincul
8318, Neuque n, Argentina.
The modern archosaur experimental subjects used in this study include four different developmental stages of Alligator mississip-


piensis from Collections of the Center for Interdisciplinary Biosciences, Technology and Innovation Park, Pavol Jozef Safárik Univer-
sity in Ko
sice, Slovak Republic. These specimens were donated to Martin Kundrát by Dr. James Perran Ross from the Department of
Wildlife Ecology and Conservation, University of Florida, Gainesville, Florida, USA.

METHOD DETAILS

Provenance of the analyzed specimen

The specimen, MCF-PVPH-847, originally represented by a complete (unprepared) egg was bought by Terry W. Manning from an
Argentinian dealer in Tucson in 2001, who is known to have collected only in Patagonia. John Nudds has the name and details of
this company (which now deals solely in European minerals) and met the dealer in Tucson in 2015 who told him that the specimen
had come from the Allen Formation of Bajo de Santa Rosa in Rı́o Negro Province. The prepared specimen was repatriated to the
Museo Municipal ‘‘Carmen Funesˮ in Plaza Huincul, Neuque n Province, Argentina, where the specimen was catalogued under
the number MCF-PVPH-874.

e1 Current Biology 30, 1–7.e1–e2, November 2, 2020

 Please cite this article in press as: Kundrát et al., Specialized Craniofacial Anatomy of a Titanosaurian Embryo from Argentina, Current Biology (2020),
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Chemical Preparation
The analyzed specimen, MCF-PVPH-874, was in ovo prepared by Terry W. Manning using a chemical method that etches away only
10 mm of the rock a day. He found that the usual concentration of 5% acetic acid was too strong, being prone to erode the very deli-
cate bones of the preserved embryo. At less aggressive concentrations of 0.5%–2.0%, however, the acid solution becomes locally
stratified, with saturated acetate solution next to the fossils inhibiting progressive action of the acid. This problem was overcome by
agitating the solution, which allowed the active acid to remain in direct contact with the exposed bone. A small quantity of wetting
agent and an algal inhibitor were added to the etching solution to maintain the rate of reaction with the specimen and prevent sub-
sequent appearance of microorganisms. At intervals during the acid treatment, digested matrix was carefully washed away, using
weak, precisely controlled jets of distilled water; even fine needles were too coarse for this stage of the process. Then the specimen
was washed in running tap water, air-dried and finally the exposed bone was protected with dilute Paraloid B72 in acetone.

Geochemistry
The whole-rock geochemistry of the samples was determined by X-ray fluorescence (XRF) techniques using a PANalytical Axios
wavelength-dispersive spectrometer (WDS) fitted with a Rhodium X-ray tube. Major element analysis was performed using the Omn-
ion package optimized for geological samples. Trace element data were obtained using the Protrace trace element package using
standard conditions. Loss on ignition (LOI) was determined at 110 C and 1100 C. Samples were analyzed as pressed powder pellets
(12 g sample, 3 g Hoechst wax, C micro powder). The samples were also analyzed by X-ray (powder) diffractometry (XRD) to identify
the principle minerals present. The XRD data were obtained using a Bruker D8 Advance diffractometer equipped with a Göbel mirror
and a Lynxeye detector. Soller slits on the X-ray tube, the antiscatter slits, were kept wide open (3 C). The diffraction patterns were
acquired using Cu Kalpha1 radiation with scans over a range of 5 -70 2 q, using a step size of 0.02 2 q and a count time of 0.2 s/step.
Patterns were matched to standards from the ICDD (International Centre for Diffraction Data) database using Eva version 14. Major
elements data is provided as weight percentages (wt %) expressed as their oxides), while trace-element concentrations are ex-
pressed in parts per million (ppm). These data reveal a number of geochemical differences.

Physical thin-sectioning
The petrographic thin sections were prepared according to the following methodology: 1) eggshell samples were embedded in
bicomponent epoxy resin (Lamit 109; Kittfort); 2) the embedded samples were ground on a Montasupal grinder (Germany) using
SiC (grain size: 400–600 nm); 3) warm reimpregnation of the ground surface with EpoFix (Struers); 4) fixation of the samples to slides
using epoxy resin (type 109); 5) fixed samples were sectioned using a diamond knife (diameter 150 mm, Struers); 6) fixed samples
were thinned on the Montasupal grinder using the abrasives of 240, 400 and 600 grits combined with ultrasound cleaning to reach a
thickness of 0.2 mm; 7) final manual abrasion using 1000 grit SiC to reach a thickness of 30 mm; and finally 8) the sections were cover-
slipped using a synthetic resin or polished on the Planopol TS (Struers).

Scanning electron microscopy

Regarding eggshell microstructure analysis, the selected samples of the specimen MCF-PVPH-874 were broken into fragments,
some of which were prepared as standard petrographic thin sections (w 30 mm) to be studied using both transmitted and polarized
light microscopy (see above). The other fragments were mounted on aluminum stubs, coated in gold (30 nm), and imaged under the
Zeiss Supra 35-VP (Carl Zeiss SMT, Oberköchen, Germany) field emission scanning electron microscope (SEM) equipped with a
VPSE detector for low vacuum conditions, a Robinson BSD for backscattered electron imaging, and coupled with an EDAX Apex
4 (Ametekh, Mahwah, USA) EDS-detector for dispersive X-ray microanalysis. Structural parameters such as shell/ layer/crystalline
unit dimensions and pore width were measured with software of the Zeiss Supra 35-VP SEM facility.

Wholemount skeletal staining

We used in toto combined Alcian Blue-Alizarin Red staining in this study following the procedure described by Wassersug [30]. Wilde-
type embryos of Alligator mississippiensis were collected under the supervision of James Perran Ross in Florida in 1999, 2001 and
2003, and are donations of the Florida Museum of Natural History and the Fish and Wildlife Conservation Commission, USA. Pho-
tographs were taken using a Spot RT camera (Diagnostic Instruments) set on a Nikon SMZ-U dissecting microscope, and were
assembled using Adobe Photoshop and CorrelDRAW X5.

3D Imaging
The in ovo preserved embryonic skull, MCF-PVPH-874, was scanned at beamline ID 19 of the European Synchrotron Radiation Fa-
cility. The scans were collected with propagation phase contrast synchrotron microtomography using a pink beam with two different
energies 71.2 KeV and 115 keV. The scanned data of the complete and a partial specimen has an isotropic voxel size of 5.06 mm,
7.46 mm and 30 mm, respectively. The reconstructed slices were converted into 16-bit .tif image stacks that were concatenated to
obtain a single stack covering the area of interest. To reduce the data size for morphological and histological observations, a second
version of the reconstructed scan was calculated with a 2x2x2 binning and an 8-bit conversion. Mimics v.12.3 and v.13.1 (Materialise
HQ, Leuven, Belgium) were used for the segmentation and 3D rendering of the skull. The internal structure and vasculature of the
premaxilla was reconstructed using VGStudio Max 2.2 (Volume Graphics, Heidelberg, Germany). The mirrored reconstruction of
the skull was rendered in Blender v.2.78 (GPL software, Blender Foundation, https://www.blender.org/).

Current Biology 30, 1–7.e1–e2, November 2, 2020 e2

Current Biology, Volume 30

Supplemental Information

Specialized Craniofacial Anatomy

of a Titanosaurian Embryo from Argentina
Martin Kundrát, Rodolfo A. Coria, Terry W. Manning, Daniel Snitting, Luis M.
Chiappe, John Nudds, and Per E. Ahlberg
Figure S1. The 3D in ovo preserved skull of a titanosaurian embryo MCF-PVPH-874. Related to Figures 1 and 4. (A) In left
lateral view. (B) In anterior view. (C) In ventral view; notice the rostral pointed projections (red arrows) of the left and right
premaxillary. (D) Close-up of the alveolar region of the right premaxilla of a titanosaurian embryo MCF-PVPH-874. Notice five
alveolar fossae and teeth (arrows) preserved in situ. Some of the fossae contain more then one alveolus. Abbreviations: alv,
alveolus; aof, antorbital fenestra; de, dentary; fr, frontal; la, lacrimal; mx, maxilla; na, nasal; or, orbit; pa, parietal; pfr, prefrontal;
pmx, premaxilla;
Figure S2. Renderred model of the 3D in ovo preserved skull of a titanosaurian embryo MCF-PVPH-874. Related to Figure
1. (A) In left lateral view. (B) In right medial view. (C) In anterior view. (D) In posterior view. Abbreviations: ag, angular; de, dentary;
epg, ectoterygoid; fr, frontal; ju, jugal; L, left; la, lacrimal; mx, maxilla; na, nasal; pa, parietal; par, prearticular; pfr, prefrontal; pg,
pterygoid; pmx, premaxilla; pob, postorbital; pt, palatine; q, quadrate; qj, quadratojugal; R, right; sag, surangular; sco, sclerotic
ossicle; sp, splenial; sq, squamosal; vo, vomer.
Figure S3. Renderred model of the 3D in ovo preserved skull of a titanosaurian embryo MCF-PVPH-874. Related to Figure
1. (A) In ventral view. (B) In dorsal view. Abbreviations: ag, angular; de, dentary; epg, ectopterygoid; fr, frontal; ju, jugal; L, left; la,
lacrimal; mx, maxilla; na, nasal; pa, parietal; pfr, prefrontal; pg, pterygoid; pmx, premaxilla; pob, postorbital; pt, palatine; q, quadrate;
qj, quadratojugal; R, right; sag, surangular; sco, sclerotic ossicle; sq, squamosal; vo, vomer.
Figure S4. Putative in ovo preserved soft tissue associated with the skull of a titanosaurian embryo MCF-PVPH-874 and
ossification of the neurocranial anatomy of the MCF-PVPH-874 titanosaurian embryo and a modern crocodilian. Related to
Figure 1. (A) Putative soft tissue is embedded in surrounding sediment and found lateral to the postero-dorsal aspect of the
embryonic parietal (arrow). (B) Rendering of three successive layers of the putative soft tissue in the same position (arrow) as
shown in (A); the associated sediment was virtually removed. (C) A transversal (coronal) projection through the critical region of the
specimen showing different texture of the parietal bone versus three layers of putative muscles that overlay the bone. (D) The
calcified braincase elements in postero-lateral view. (E) Successive ossification stages of braincase elements of Alligator
mississippiensis in ventral view. (F) A transversal (coronal) projection through the brainces region of the specimen showing different
texture of cartilaginous and calcified tissues. Abbreviations: bo, basioccipital; cp, cultriform process of the parabasisphenoid; fm,
foramen magnum; ls, laterosphenoid; pbs, parabasisphenoid; pro+op, prootic+opisthotic; scc, semicircular canal; so, supraoccipital.