SUBJECT AREAS:
PHYLOGENETICS
EMBRYOLOGY
GEOCHEMISTRY
PALAEONTOLOGY
Filling the gaps of dinosaur eggshell
phylogeny: Late Jurassic Theropod clutch
with embryos from Portugal
Ricardo Araújo1,2, Rui Castanhinha2,3, Rui M. S. Martins2,4,5,6, Octávio Mateus2,7, Christophe Hendrickx2,7,
F. Beckmann8, N. Schell8 & L. C. Alves4,6
1
Received
3 December 2012
Accepted
2 May 2013
Published
30 May 2013
Correspondence and
requests for materials
should be addressed to
R.A. (rmaraujo@smu.
edu)
Huffington Department of Earth Sciences, Southern Methodist University, Dallas, Texas, 75275-0395, 2Museu da Lourinhã, Rua
João Luis de Moura, 95, 2530-158 Lourinhã, Portugal, 3Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6P-2780-156
Oeiras, Organogenesis Ibn Batuta (A1) - Room 1A, Portugal, 4IST/CTN, Campus Tecnológico e Nuclear, EN10, 2696-953
Sacavém, Portugal, 5CENIMAT/I3N, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Quinta da Torre,
2829-516 Caparica, Portugal, 6Centro de Fı́sica Nuclear da Universidade de Lisboa (CFNUL), Av. Prof. Gama Pinto 2, 1649-003
Lisboa, Portugal, 7CICEGe, Centro de Investigação em Ciência e Engenharia Geológica, Faculdade de Ciências e Tecnologia da
Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal, 8Helmholtz-Zentrum Geesthacht (HZG), MaxPlanck-Str. 1, 21502 Geesthacht, Germany.
The non-avian saurischians that have associated eggshells and embryos are represented only by the
sauropodomorph Massospondylus and Coelurosauria (derived theropods), thus missing the basal theropod
representatives. We report a dinosaur clutch containing several crushed eggs and embryonic material
ascribed to the megalosaurid theropod Torvosaurus. It represents the first associated eggshells and embryos
of megalosauroids, thus filling an important phylogenetic gap between two distantly related groups of
saurischians. These fossils represent the only unequivocal basal theropod embryos found to date. The
assemblage was found in early Tithonian fluvial overbank deposits of the Lourinhã Formation in West
Portugal. The morphological, microstructural and chemical characterization results of the eggshell
fragments indicate very mild diagenesis. Furthermore, these fossils allow unambiguous association of basal
theropod osteology with a specific and unique new eggshell morphology.
T
he discovery of fossilized eggs and embryos together is rare1–3. Eggshells associated with non-avian saurischian embryos are reported for (i) the Early Jurassic sauropodomorph Massospondylus4, (ii) the Late
Jurassic avetheropod Lourinhanosaurus5,6, (iii) a Late Cretaceous therizinosauroid7, (iv) Early and Late
Cretaceous titanosaurs8, (v) the Late Cretaceous troodontids9,10, and (vi) a Late Cretaceous oviraptorid11. Thus,
there is a dearth of knowledge regarding eggs associated with embryos at the base of Saurischia, especially prior to
the Late Cretaceous. Fossil embryos provide important information about (i) ontogenetic transformations including heterochronic processes12, (ii) developmental pathways in multiple lineages13, (iii) reproductive behaviors
including parental care14, and (iv) the evolution of physiological regimes15. Yet, studies on embryo osteology are
particularly difficult because species-level identification is often hampered by poor ossification of the specimens,
which can result in a paucity of diagnostic anatomical features11. The purpose of this paper is to link a new eggshell
morphology to the osteology of a particular group of basal theropod dinosaurs, as well as to evaluate the extension
of diagenesis on the morphology of the eggshell. We employed synchrotron radiation-based micro-computed
tomography (SR-mCT), scanning electron microscopy (SEM), and optical microscopy techniques to study the
morphology and microstructure of the eggshell fragments (see Methods). Additionally, eggshells and surrounding
sediment characterization was performed by using an array of complementary techniques in order to assess the
extension of diagenesis, namely micro-Proton Induced X-ray Emission (micro-PIXE), Synchrotron radiationbased X-Ray Diffraction (SR-XRD) and cathodoluminescence (CL) (see Methods and Supplementary Notes).
Results
Discovery. The specimen (ML1188, ML – Museu da Lourinhã, Portugal) is composed of a large number (. 500)
of clustered eggshell fragments forming an assemblage 65 cm in diameter, containing embryonic bones and teeth
(Fig. 1; Supplementary Note 1). No other vertebrate remains or eggshell fragments were found in the immediate
area. The assemblage was discovered on a grey mudstone layer (39u 139 N; 9u 209 W; exact coordinates available
SCIENTIFIC REPORTS | 3 : 1924 | DOI: 10.1038/srep01924
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Figure 1 | (a) Right maxilla, dentary and jugal of Torvosaurus sp. (ML 1188) in medial view. (b & c) Anterior part of right maxilla of Torvosaurus sp. (ML
1188) in medial view. (d) Right dentary of Torvosaurus sp. (ML 1188) in medial view. (e) Anteriormost maxillary teeth in medial view. (d) Anteriormost
dentary teeth in medial view. Abbreviations: amp, anteromedial process; anr, anterior ramus; aof, antorbital fenestra; cx, cervix dentis; d, dentary; d1,
isolated first dentary tooth; d2, second dentary tooth; d3, third dentary tooth; d5, fifth dentary tooth; dt7, seventh dentary tooth; dca, distal carina; idp,
interdental plate; j, jugal; jur, jugal ramus; lac, lacrimal contact of the maxilla; m, maxilla; mca, mesial carina; mf, Meckelian fossa; mfo, Meckelian
foramina; mg, Meckelian groove; ms, mandibular symphysis; mx1,first maxillary tooth; mx2, second maxillary tooth; mx5, fifth maxillary tooth; nug,
nutrient groove; pdg, paradental groove; ro, root. Scale: 10 mm for (a), 5 mm for (b), (c) & (d); 2 mm for (e) & (f).
upon request) in August 2005 by A. Walen at Porto das Barcas in the
Sobral Member of the Lourinhã Formation. Excavation was
performed in September 2005 and May 2006. A single block
containing the entire assemblage was jacketed in the field using a
specific technique previously described16. The specimen was
prepared at the Museu da Lourinhã in 2009. Another clutch
(ML565) previously described5, referred to as the Paimogo nest,
was found in penecontemporaneous deposits of the Lourinhã
Formation in 1993 by I. Mateus, less than 10 km away from the
area where ML1188 was collected. The Paimogo nest was
recovered from an iron-rich mudstone layer. Despite the dubious
phylogenetic position of the avetheropod Lourinhanosaurus
antunesi6,17, the Paimogo nest was tentatively ascribed to this taxon.
Clutch. In spite of post-burial compression18, the observation of
continuous patches of eggshells allows discerning eggshell
orientations, which indicate the presence of different eggs. In
Supplementary Note 1a the assemblage can be divided into three
different mounds: one at the top, one at the middle, and one at the
bottom. The top mound has eggshell patches with N-S fracture
orientation, whereas the middle and lower mounds E-W fracture
patterns. All the eggs are heavily crushed, and individual boundaries between the eggs are difficult to distinguish (Supplementary
Note 1). Nevertheless, this assemblage forms a clutch (ML1188)
because it consists of several eggs (. 3) mingled with embryonic
bones. There are generally one or two eggshells vertically stacked,
but rarely three or four eggshells are found piled up (Supplementary
Note 1). Embryonic material in the clutch consists of five dispersed
and isolated teeth, a maxilla preserving four teeth, a dentary with four
teeth in situ, an isolated dentary tooth, a series of three articulated
centra, and other unidentifiable bones (Fig. 1; Supplementary Note 1,
2, 4, and 6). ML1188 was found as an isolated and concentrated
clutch without any surrounding dispersed eggshells.
Embryonic remains. The medial side of the incomplete right maxilla
is on the surface of the clutch (Fig. 1, Supplementary Note 2).
The partial maxilla comprises the anterior ramus, an incomplete
SCIENTIFIC REPORTS | 3 : 1924 | DOI: 10.1038/srep01924
ascending process, and the jugal ramus whose the posterior part is
separated from the rest of the maxillary bone. The main body has a
Subtriangular anterior ramus projecting moderately anteriorly from
the antorbital fenestra (Fig. 1). The anterior margin of the anterior
ramus is convex and forms a right angle with the ventral margin of
the maxilla. The anterodorsal margin of the anterior ramus is slightly
concave and confluent with the ascending process, thus, there is no
step delimiting the two structures. The jugal ramus is broken in two
pieces, the posterior part lying one centimeter below the maxilla.
Once digitally restored (Supplementary Note 3), the jugal ramus is
elongated with some parts in the middle of the ramus missing. The
dorsal margin of the horizontal ramus, corresponding to the ventral
rim of the antorbital fenestra, is straight and subparallel to the tooth
row in its anterior part whereas the posterior part of the ramus has a
sigmoid outline and slopes ventroposteriorly towards the jugal
contact of the ramus. The posteriormost part of the jugal ramus
corresponds to a tongue-like process delimited by a small concavity on its dorsal margin and the main axis passing through this
posterior process is oriented ventroposteriorly. The ventral margin of
the jugal ramus is straight and parallel to the anteroposterior axis of
the jugal process. Little information can be extracted from the jugal
but the contact between the jugal and maxilla extends one third on
the lateral side of the jugal ramus. The articular surface for the
lacrimal is visible on the anteromedial part of the jugal, at the level
of the posterior process of the jugal ramus. The lacrimal was also
contacting the maxilla along one third of the jugal ramus. The
ascending process is thick at its base and tapers dorsally. The
anteroventral margin of the ascending process is convex, almost
forming an obtuse angle, and the main axis passing through this
process angles 28u with the ventral margin of the maxilla. Two
elongated and subparallel ridges are present at the dorsal tip of the
ascending process and delimit the lacrimal contact of the maxilla.
The antorbital fenestra is parabolic in outline. There is no medial
antorbital fossa. The medial surface of the maxilla bears parallel
stripes corresponding to vascular canals. On the main body of the
maxilla, these canals are anteroposterioly aligned but, on the
ascending process they are parallel to its main axis. However,
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the canals are more irregularly placed on the anterior part of the
ascending ramus. Although the anteromedial surface of the maxilla
is damaged, the surface is composed of solid bone, thus no maxillary
and promaxillary fenestrae, maxillary antrum and pneumatic
excavations are present. At the level of the teeth roots, only one
isolated interdental plate has been preserved in between the second
and fourth maxillary teeth. This suggests that the interdental plates of
the maxilla were unfused. The plate is subrectangular, and its surface
is punctuated. The anteromedial process has been crushed at the level
of the anterior margin of the maxilla. The anteromedial process is
located on the anterodorsal border of the anterior ramus, being a long
finger-like projection parallel to the anterodorsal rim of the anterior
ramus. Four maxillary teeth are preserved on the anterior part of the
maxillary body and there is an isolated tooth near the maxilla and two
more loose teeth located some distance away from this bone. The
maxillary tooth count is difficult to estimate because the posterior
part of the main body bears no teeth. However, on the anterior ramus
of the maxilla there were probably five teeth. All teeth are strongly
elongated (Crown Height Ratio . 2.5), their apices are sharply acute
and both mesial and distal carinae lack serrations. The mesial and
distal profiles are recurved distally and the crowns are subconical.
Nevertheless, the lingual surface of the crowns is mostly flattened
except the basal surface which is concave. This concave area on the
medial surface of the crown received the erupting tooth of the
maxilla. The enamel surface of the teeth is smooth and does not
bear transversal undulations, enamel wrinkling, longitudinal
grooves, or wear facets.
A right dentary is lying on its lateral side just below the anterior
part of the maxilla (Fig. 1, Supplementary Note 4). The bone is almost
complete and only part of the bone posterior to the opening of the
Meckelian fossa is missing. It is fractured in the middle by a transversal fissure. The dentary is massive, with a ventrodorsally large
medial wall remaining the same width along the bone. Four fully
erupted teeth are present on the dentary but only two of them, the
third and seventh dentary teeth, are complete. A fifth isolated tooth,
most likely the first dentary tooth, is lying beside the anteroventral
corner of the dentary. Based on the positions of the remaining teeth,
we estimate a total of eight alveoli on this portion of the dentary. The
interdental plates are unfused. Three interdental plates are preserved
in between the first and second dentary teeth, the second and third,
and the fifth and seventh teeth. Variation occurs in the shape of the
interdental plates along the tooth row as the two anteriormost plates
form a vertical rectangle whereas the third one, more posteriorly
located, has a horizontal rectangular outline. The mandibular symphysis is short, smooth and forms an elongated triangle on the anteriormost part of the dentary. The paradental groove separating the
medial wall of the dentary with the interdental plates is large, gently
concave and seems to have been open in between those two structures. The Meckelian groove is well visible on the dentary, running
along the bone just above the dorsal margin of the dentary. This
longitudinal groove is filled with sediment and seems to extend anteriorly until the third dentary teeth. The step between the Meckelian
groove and Meckelian fossa occurs at the level of the seventh dentary
tooth. Only the anterior part of the Meckelian fossa is preserved. A
large concavity at the level of the third dentary tooth is present just
above the ventral margin of the dentary and includes a short anteroposterioly oriented ridge. Two small grooves located in between
this concavity and the mandibular symphysis are interpreted to be
Meckelian foramina (Fig. 1). The anteroventral margin of the dentary is rounded and almost subrectangular, with the inflexion point
close to the level of the anteriormost point of the dentary. The anteroventral margin is unexpanded with no articular brace forming a
chin, and the ventral margin appears to have been fairly straight. The
teeth of the dentary are unserrated and the crowns are pointed,
strongly basoapically elongated and recurved posteriorly, and devoid
of enamel structure.
SCIENTIFIC REPORTS | 3 : 1924 | DOI: 10.1038/srep01924
Three articulated amphiplatyan vertebrae were fully prepared
(Supplementary Note 6). The ventral part of the centrum bears
two paired pits identified as neurovascular foramina. The anterior
and posterior faces of the vertebrae show evenly distributed small
pits, consistent with an early developmental stage8. The centrum
faces are expanded (, 38% relative to the mid-centrum) and bear
confluent striations, whereas in the median part of the centrum, the
striations are parallel. Although other fragmentary bones are present
and scattered within the clutch none are identifiable, thus, no further
information could be obtained.
Eggshell characterization. The total eggshell thickness of the ML1188
eggshells is approximately 1.2 mm. They bear (i) anastomizing
ornamentation resembling to some degree the patterns of linearituberculate1 ornamentation, consisting of sub-circular to subelliptical grooves separated by interconnected sharp ridges (Fig. 2c,
3a), (ii) acicular, elongated blade-shaped, calcite crystals radiating
from the base of the mammillae to the outermost part of the
eggshell (Fig. 2b), and (iii) only one primary layer (Figs. 2a,b, 3a,b).
Growth lines are visible across the entire thickness of the eggshells,
but tend to be concentrated at the base and develop perpendicularly to
the acicular calcite crystals of the wedges (Fig. 2b). The mammillae
height, which is difficult to measure for this eggshell morphotype,
averages 166 mm, corresponding to about 17% of the total thickness
of the eggshell (Figs. 2b, 3b).
The results presented in Fig. 3 show that SR-mCT is a useful technique for non-destructive imaging of the eggshell morphology. The
morphology of the pores is provided by high-quality data that is
difficult to visualize by SEM or thin-sections. The pores are irregular
canals that ramify and vary in width along their length (i.e., resembling prolatocanaliculate1). Their diameter is highly variable because
the contour is irregular, ranging between , 100 mm and 500 mm.
Pores anastomose with adjacent pores close to the outer surface
(Fig. 2, 3). In fact, the pores seem to form an interconnected system
(Fig. 3c and Supplementary videos). All eggshells have equivalent
pore density, which indicates that all the eggs were buried under a
homogenous incubating medium19. Eggshell ornamentation consists
of well-defined ridges and islets with prominent and sub-vertical
walls, which show no flattening or smoothing of their sharp edges
Figure 2 | ML1188 eggshell (a) SEM micrograph of the eggshell radial
section (b) optical micrograph eggshell radial section showing acicular
crystals and a single layer; (c) SEM micrograph of the external surface of
the eggshell, and (d) SEM micrograph of the internal surface of the
eggshell.
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(Fig. 4). Furthermore, SR-XRD (Fig. 4) and micro-PIXE (Supplementary Note 8) data indicate that pore-filling sediment is composed
mainly of phyllosilicates. SR-XRD shows that the major compound
of the eggshell is calcite (Fig. 4). The inner surface shows some degree
of flattening, and in some points partial fusion of the mammillae (see
upper right corner of Fig. 2d). The eggshells do not luminesce under
the impact of accelerated electrons by the CL equipment (Fig. 5).
Luminescence is only observed on the pores. The micro-PIXE analysis of the eggshell (excluding pores) revealed the presence of Mg, Fe,
Mn, Si (0.33%, 0.27%, 0.18% and 0.10%, respectively) and several
trace elements (Na, Al, S, K and Cl), with a corresponding loss of Ca
(Table 1, Supplementary Note 8).
Figure 3 | SR-mCT image of the eggshell in (a) external, (b) internal and
(c) transverse view.
Discussion
ML1188 can be assigned to Theropoda due to the presence of
strongly elongated, sharply pointed crowns with a distal curvature.
Among non-theropod dinosaurs, this condition is only present in the
basalmost sauropodomorph Eoraptor20. Nonetheless, some Eoraptor
crowns are lanceolate and the maxillary teeth are not so elongated,
contrarily to ML1188. Among Theropoda, the antorbital tooth row
indicates tetanuran relationships17 for ML1188. An anteriorly
inclined nutrient groove between the medial wall of the maxillary
body and the interdental plate, as well as strongly convergent ventral
and dorsal margins of the jugal ramus are also two synapomorphies
for Megalosauridae (Supplementary Note 3 and 7). We ascribe
ML1188 to Megalosaurinae based on the presence of less than ten
dentary teeth, from the anteriormost point of the mandibular symphysis to the anteriormost point of the Meckelian fossa, as well as the
absence of the internal antorbital fossa on the medial side of the
maxilla (Supplementary Note 3, 5 and 7). The maxillary fenestra,
promaxillary fenestra and pneumatic recesses appear early in ontogeny and in the maxilla of Tetanurae embryos9,21. The maxillary
fenestra pierces the maxilla in most tetanurans22, but corresponds
(Fig. 3a). An external zone (Supplementary Note 1) resembling an
additional layer may be present in the eggshells. However, the SRXRD analysis of the external and internal surface zones of the
eggshell fragments shows the presence of quartz and phyllosilicates
Figure 4 | SR-XRD patterns obtained for an eggshell from ML1188 at the
topmost zone (external surface zone), at the middle and at the
bottommost zone (internal surface zone) of the eggshell. The data were
acquired in transmission mode (beam closely parallel to the outer/inner
surfaces of the eggshell). The identification of the compounds present in
each zone was performed by comparing the measured SR-XRD patterns
with data sets from the PDF-2 database.
SCIENTIFIC REPORTS | 3 : 1924 | DOI: 10.1038/srep01924
Figure 5 | Radial section of the eggshell under (a) polarized lens and (b)
cathodoluminescence. There is no part of the eggshell that luminesces
except on the pores due to the presence of phyllosilicates.
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Table 1 | Compositional results for the eggshell and pore inclusions
as determined by micro-PIXE technique. The remaining percentage
includes oxygen and carbon
%w
eggshell
pore inclusions
Na
Mg
Al
Si
P
S
Cl
K
Ca
Ti
Mn
Fe
0.09
0.33
0.09
0.10
n.d.
0.08
0.02
0.03
39.4
n.d.
0.18
0.27
0.6
1.9
11.6
25.2
0.07
0.09
0.08
2.4
6.0
0.4
0.03
3.6
to a small opening located within the internal antorbital fossa
in some basal tetanurans22,23, for example, Duriavenator and
Megalosaurus (Supplementary Note 3). However, as in ML1188, an
unfenestrated and unpneumatized maxilla lacking a medial-antorbital fossa is only present in few megalosaurids (e.g., Torvosaurus,
Dubreuillosaurus, see Supplementary Note 3). The embryonic remains of
ML1188 share three synapomorphies with both Megalosaurinae
Megalosaurus and Torvosaurus: a blunt and unexpanded anteroventral margin of the dentary, very elongated maxillary crowns (Crown
Height Ratio . 2.5) and tall and unfused anteriormost interdental
plates of the dentary (Supplementary Note 3, 5 and 7). This embryo
can be referred to the genus Torvosaurus due to the lack of pneumaticity posterior to the base of the ascending process, an angle of
less than 35u in between the base of the ascending process and the
ventral margin of the maxilla, and the tongue-shaped extremity of
the jugal ramus of the maxilla. The maxilla of Megalosaurus displays
two pneumatic excavations on the medial surface of the maxillary
body, dorsal to the lingual bar and posterior to the ascending process
in medial view23. In addition, the angle in between the base of the
ascending process and the alveolar margin of the maxilla exceeds 35u
and the posterior extremity of the jugal ramus tapers as a very long
pointing process. Importantly, Torvosaurus has been previously
reported from the same Formation in Portugal24. Four features differentiate ML1188 from the maxilla of adult Torvosaurus: (i) absence
of serrations on the mesial and distal carinae, (ii) absence of fusion of
the interdental plates in the maxilla, and an (iii) anteroposteriorly
short anterior ramus (iv) bearing less than six maxillary teeth. These
features are most likely related to morphological variation through
ontogeny. The lack of tooth serrations among non-coelurosaur theropods is rare, being only observed in teeth of Spinosaurinae25.
However, the lack of denticles in portions of the maxillary teeth
has been observed in the embryo of the basal avetheropod
Lourinhanosaurus ML565-122, namely in the mesial and apical portion of the distal carinae. Likewise, the embryonic specimen of
Troodon also bears unserrated crowns, in contrast to the condition
found in adults9. ML1188 is the only case of complete absence of
serrations known to date in non-coelurosaur theropod embryos. In
coelurosaurs, besides Troodon, the Byronosaurus embryo also lacks
serrations as in the adult9,10. Separated interdental plates occur in the
maxilla in several adult specimens of megalosaurids (e.g., Megalosaurus, Duriavenator). In the Torvosaurus holotype (BYUVP 9122,
BYUVP - Brigham Young University Vertebrate Paleontology,
Provo, Utah, USA), and a larger maxilla (ML1100) discovered in
the Lourinhã Formation, the maxillary interdental plates are completely fused unlike part of the dentary in the holotype of
Torvosaurus (BYUVP 2003). Unpreserved interdental plates in the
hatchling Allosaurus21 may suggest that these elements were unfused
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in juveniles, contrary to the condition seen in adult Allosaurus. Thus,
fusion of interdental plates in the maxilla of basal tetanurans seems to
occur after hatchling. Likewise, the proportion of the anterior ramus
and the maxillary tooth count have also been previously described as
traits that may vary ontogenetically at post-embryonic stages21.
These four conditions, the absence of denticles, fusion of the maxillary interdental plates, elongation of the anterior ramus and the
differences in maxillary tooth counts, are traits that vary trough
theropod ontogeny after hatchling.
Preservation of the eggshells ranges from pristine to slightly
abraded mammillae, corresponding to Stage 1 of eggshell corrosion26,
and the eggshells show little evidence of bacterial or chemical corrosion26,27. The minor flattening of the mammillae can be due to
bacterial action, although natural degradation of the eggshell can produce similar results27. Furthermore, no corrosion pits or pinholes are
present. Other manifestations of very mild diagenesis are the absence
of: epitaxial calcite growth blankets, "ghost" crystals, and criss-crossed
calcite veins17,29. At the micrometer scale, individual calcite grains are
distinct and show neither smoothing nor the fibrous texture that has
been detected in acidic and/or high temperature conditions in avian
eggshells26. The external ornamentation of the ML1188 eggshells is
distinct from the honeycombed surficial pattern caused by acidic corrosion. The pattern of thin, sharp, straight walls visible in heavily
corroded eggshells26 differs from the broad, meandering and anastomosing external ornamentation of ML1188 eggshells. The putative additional layer detected in some shells (see Results) is due to sediment
from the surrounding matrix and does not represent a true secondary
layer15. In fact, this layer is composed of extrinsic material not pertaining to the eggshell (Fig. 4 and 5, Table 1, Supplementary Note 8 and 9).
Thus, from an external morphology perspective there is no evidence of
diagenesis. However, diagenesis can affect the eggshell at a chemical
level. The non-luminescence of the eggshells (Fig. 5) could imply that
there was little to no diagenetic influence on the eggshells. Nevertheless, the quenching effect of Fe21 has to be taken into consideration29,30 as it may mask the extension of diagenesis. In fact, micro-PIXE
revealed trace amounts of Fe as well as other Ca-substituting elements
(see Results). Therefore, the minimal presence of these elements and
corresponding low loss of Ca in calcite, indicates very mild diagenesis.
On the other hand, the luminescence observed on the pores (Fig. 5) is
due to the presence of phyllosilicates, which are rich in luminescent
elements. The combination of CL and micro-PIXE techniques is thus
necessarily complementary. This series of tests indicate that eggshell
external morphology has not been altered by diagenetic effects.
Diagenesis affected only, very mildly, at a chemical level the eggshell’s
composition.
The clutch was not significantly taphonomically disturbed (Supplementary Note 1 and 6). There are several facts supporting this
statement, namely: (i) the presence of articulated vertebrae, (ii) the
concentrated clutch without surrounding dispersed eggshells, (iii)
the undisplaced eggshells within the clutch, and (iv) the poorly
cemented teeth found in situ in the maxilla and dentary, (v) the nest
was located in a low energy fluvial overbank setting. However, the
preservation of few bone elements seems to be contradictory
evidence. This can be explained by the reduced mineralization of
embryonic bones, which results in increased degradation and
decomposition. This nearly undisturbed taphonomic scenario can
be explained by deliberate burial from the progenitor, analogously to
the behavior seen in extant seaturtles. Importantly, the eggshells are
highly porous. Porous eggshells have high gas and vapor conductance, which allows efficient gaseous exchange between the external
and internal media, whereas simple porous systems are indicative of
exposed nesting conditions31. The eggshells are highly porous and,
thus, indicative of eggs buried for incubation within the substrate.
ML1188 could have been buried by the progenitor because of the
high porosity of the eggshells, undisturbed taphonomic setting, and
low-energy geological context.
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ML1188 eggshells allow ascription to a non-maniraptoran
saurischian, based on the derived characters from a previous
phylogenetic optimization of egg characters2: (i) presence of surficial
ornamentation, (ii) acicular crystals as building blocks of the eggshell
structure, and (iii) absence of two aprismatic layers. What was previously thought to be a theropod synapomorphy concerning the tubular shape of the pore canals32, does not hold true considering the
prolatocanaliculate-type1 pores in ML1188. Using parataxonomy,
it is difficult to determine whether these eggshells belong to the
dinosauroid-spherulitic basic shell type, due to the presence of distinct shell units and boundaries. The ML1188 eggshells are most
closely related to the Dendroolithidae oofamily based on the presence of (i) a single eggshell layer, (ii) irregularly shaped shell units,
(iii) branching fans, (iv) lack of true mammillae, (v) pore canals
forming an intercone space, (vi) prolatocanaliculate type1,33, and
(vii) shell units extending to the surface (prismatic condition).
Dendroolithid eggshells have only been reported from the Late
Cretaceous of China and Mongolia33,34, thus, ML1188 represents
the first shells of this type known from the Late Jurassic.
Dendroolithidae eggshells were thought to be associated with either
sauropods or ornithopods35, but the ML1188 embryos definitively
link this type of eggshell to theropods (Fig. 1–3).
Concerning non-theropod taxa, titanosaurid eggshells are composed of one structural layer, whereas. ML1188 eggshells (1.2 mm)
are slightly thinner than the 1.8 mm titanosaurid eggshells37, but the
external ornamentation is superficially similar. SEM imaging, optical
microscopy and SR-mCT analysis reveals an intricate anastomosing
pattern in ML1188 (Fig. 2, 3) that is distinct from the nodular
surficial pattern of Titanosauridae eggshells28. Furthermore, ultrastructural features reveal a very different pore system and arrangement of shell units. Titanosauridae eggshells have broad open-angled
shell units28, in contrast to ML1188 eggshell units that form a sharp
angle where they meet the outer surface, producing its peculiar ornamentation (Supplementary Videos). Also readily excludable are
ornithischian eggshells: Hypacrosaurus sternbergi eggshells do not
have individualized shell units28, and the neoceratopsid IGM 100/
2010 (IGM - Institute of Geology, Ulaan Bataar, Mongolia) possesses
several eggshell layers and blade-shaped crystals at the base of the
eggshell13.
Apart from the ML1188 embryos, there are no other reported
megalosauroid embryos. This finding not only identifies a unique
and previously unknown eggshell morphotype, but more importantly, it bridges a phylogenetic gap at the base of the Theropoda
clade (Fig. 6). Nevertheless, it should be noted that the single eggshell
layer and acicular morphology are Saurischia synapomorphies2.
ML1188 eggshells represent the first report of theropod eggs with a
single structural layer, the plesiomorphic condition for Theropoda.
The two non-coelurosauran tetanuran taxa, Lourinhanosaurus
Figure 6 | The known record of embryos and associated eggshell structure explicits the phylogenetic position of the Torvosaurus embryos (ML1188),
which occupies a gap at the base of the Theropoda cladogram. Dashed lines indicate the dubious position of Lourinhanosaurus as an Allosauroidea or as a
basal Coelurosauria in the light of the most recent analysis17. Major clades in bold indicate the presence of associated embryo-eggshell fossils. For
attribution information on the individual elements of this figure please see the Supplementary Information.
SCIENTIFIC REPORTS | 3 : 1924 | DOI: 10.1038/srep01924
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antunesi ML565 and Torvosaurus ML1188, have clearly distinct eggshell morphotypes. ML1188 eggshells differ from those tentatively
ascribed to Lourinhanosaurus antunesi because the later (i) lack
ornamentation, (ii) exhibit a tubular and oblique pore system, (iii)
are thinner (average 5 0.92 mm)36. Moreover, eggshells assigned to
Lourinhanosaurus antunesi bear two distinct layers, and the basal
layer displays large nucleation centers18. Although the ML1188
embryos and those assigned to Lourinhanosaurus antunesi are morphologically similar and closely related phylogenetically, they have
strikingly different eggshell morphologies. The marked difference in
the eggshells of these two taxa implies that the taxonomic assignment
of ML565 to Lourinhanosaurus antunesi can be either incorrect, or
eggshell characters are highly homoplastic in basal theropods, or the
phylogenetic position of Lourinhanosaurus antunesi is uncertain,
probably being an Allosauroidea or basal Coelurosauria17. Ongoing
research on the ML565 embryos is attempting to resolve this issue.
ML1188 eggshells are different from those of the coelurosaur
Troodon formosus. The later, with an average thickness of
1.02 mm, exhibit (i) two structural layers, (ii) pores with a constant
diameter, and (iii) spherulites composed of blade-like calcite crystals.
The other coelurosaur theropods can be readily excluded because
oviraptosaurids and ML565 are composed of two structural layers,
and Avialae eggshells have three layers2.
Methods
Geology. The Lourinhã Formation is located on the west coast of Portugal in the
Lusitanian Basin, a rift structure that formed during the opening of the proto-North
Atlantic in the Jurassic38. The Upper Jurassic Lourinhã Formation was deposited
during a period of global sea level rise and concomitant local subsidence39. The
resultant increase in accommodation space facilitated development of coastal
meandering fluvial and delta fan environments punctuated by brief marine
incursions40. Abundant dinosaur bones and tracks, as well as fossil remains of other
continental vertebrates, are an important component of the fauna in this formation40.
The Sobral Member of the Lourinhã Formation is composed of deltaic sandstones and
mudstones41 (Supplementary Note 10). The Sobral Member is early Tithonian in age,
based on stratigraphic correlation, vertebrate and invertebrate (chiefly bivalves and
gastropods) biostratigraphy, and Sr-isotope chemostratigraphy using oyster
shells41,42. The layer from which the clutch was collected is approximately 35 m above
the Kimmeridgian-Tithonian boundary41 (152.1 6 0.9 Ma43) (Supplementary Note
10).
Eggshell characterization. An analysis of the general morphology, microstructure,
and elemental composition of fossil eggshell as well as the identification of the
compounds present is necessary to provide pertinent information about the extent
of diagenesis affecting external morphology and the nest environment in which
eggshells are preserved. Optical microscopy was employed to study thin-sections
sliced radially and transversely from several eggshell fragments. The observations
were performed using a Zeiss Axioplan 2 polarized light microscope and a coupled
Nikon DXM200F digital camera. For the preparation of the samples, eggshell
fragments were placed on a silicone or clay mould that was subsequently filled with
epoxy glue. The hardened block was then glued with epoxy to a microscope slide,
sectioned using a diamond disk Struers Discoplan-TS, and subsequently abraded
with fine sandpaper until the thickness allowed light to be transmitted. Finally, the
sample was polished with a Buehler PETROPOL until thin-sections reached an
optimized thickness for analysis on the polarized lens microscope. Furthermore, a
lumic HC3-LM cathodoluminescence microscope was used to study the
luminescence of the polished thin section surfaces irradiated by an electron beam
(the microscope is equipped with a tungsten filament). SEM was also used to
characterize the eggshell fragments. Observations were carried out on a JEOL JSMT330A microscope using an accelerating voltage of 20 kV. For this purpose, the
samples were cleaned in an ultrasonic bath for 5 min and subsequently coated with
gold. Cross-sectional SEM micrographs and micrographs of the outer and inner
surfaces of the eggshell were collected. SR-mCT studies were performed at the
beamline HARWI II operated by the Helmholtz-Zentrum Geesthacht (HZG) at the
storage ring DORIS III at the Deutsches Elektronen-Synchrotron (DESY) in
Hamburg, Germany. The samples were imaged in absorption mode with photon
energy of 37 keV. For acquiring the X-ray attenuation projections, the sample was
rotated between -180u and 180u in equidistant steps of 0.2u. Technical details of the
beamline are described elsewhere44. For the evaluation of the SR-mCT data, slices
perpendicular to the rotation axis were reconstructed from the single projections by
a tomographic reconstruction algorithm using ‘‘filtered backprojection’’. The slices
were then collected in an image stack that can be visualized, edited and exported
into different file formats using a three-dimensional rendering software.
Visualization of the morphology of the samples was conducted using VGStudio
Max 1.2.1 (Volume Graphics, Heidelberg, Germany) and TOMO-GPU (Project
PTDC/EIA-EIA/102579/2008). The effective pixel size corresponds to 6.4 mm. The
SCIENTIFIC REPORTS | 3 : 1924 | DOI: 10.1038/srep01924
High Energy Materials Science beamline (HEMS)45 at the storage ring PETRA III at
DESY offers the opportunity to investigate fine structural details (high flux in
parallel beam to increase resolution and enough signal in the small gauge volumes).
Eggshell fragments were broken into pieces of approximately 5 3 5 mm in order to
obtain samples exhibiting ‘‘fresh’’ cross-sections. SR-XRD data were acquired at
HEMS in transmission mode (with an image plate MAR345) using a beam spot of
100 mm in vertical and 300 mm in horizontal with 87 keV energy (the eggshell
fragments were mounted on the sample holder with the outer and inner shell
surfaces nearly parallel to beam). The samples were measured starting from the
outer surface to the inner surface of the shell, along a ‘‘line’’ (width 5 300 mm)
perpendicular to these surfaces, every 100 mm with an acquisition time of 1 s. The
compounds present in each zone were identified by comparing the experimental
data with data sets from a Powder Diffraction File database (PDF-2) maintained by
the International Center for Diffraction Data (ICCD). Micro-PIXE analysis was
used to identify and map the elemental composition of eggshell samples within
micron-scale regions. The experiments were performed at IST/CTN – Campus
Tecnológico e Nuclear, Portugal. The Ion Beam Laboratory is equipped with a 2.5
MV Van de Graaff accelerator and an ion microprobe (Oxford Microbeams) with
focused beam providing approximately 3 mm spatial resolution. The scanning
nuclear microprobe was used to obtain two-dimensional X-ray elemental
distribution maps in cross-section analysis of eggshell fragments. During the
experiments the sample was exposed to a proton beam (an area of 1860 3
1860 mm) with an energy of 1 MeV, which provided information about the shell
and pores composition.
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Acknowledgements
We would like to thank Aart Walen, who discovered the eggshell assemblage, and Verónica
Duarte as well as Vasco Ribeiro, who did most of the preparation. We thank Alexandra
Tomás and the volunteers that participated in the excavations. For the laboratorial work
(SEM and thin-sections), we are most grateful to Professor João Pais, who taught us how to
master the SEM, and also to Maria Eduarda Ferreira, Fátima Silva, and Carlos Galhano.
Irina Sandu assisted us during the thin-sections microscopic photography. We thank Simão
Mateus for drawing the maxilla illustration. We also would like to thank Professor Maria
Helena Couto from Departamento de Geociências, Ambiente e Ordenamento do
Território, Faculdade de Ciências, Universidade do Porto for assisting with the CL imaging.
Thanks are also due to Carlos Natário for fruitful discussions, particularly his insightful
discussions about the Lourinhã Formation. Louis L. Jacobs and T. Scott Myers kindly
perused the manuscript before submission. We acknowledge the use of TaxonSearch and
Phylopic. We also would like to acknowledge the financial support from the Jurassic
Foundation under the project ’Dinosaur Eggs and Embryos of the Lourinhã Formation
(Upper Jurassic, Portugal)’, from the ‘‘Fundação para a Ciência e a Tecnologia (FCT/MCE)’’
through the PTDC/BIA-EVF/113222/2009 project and from DESY through the I-20110184
EC and I-20110229 EC projects. This work has been supported by the European
Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement nu
312284. R.M.S. Martins gratefully acknowledges the financial support from FCT/MCE to
CFNUL (PEst-OE/FIS/UI0275/2011). Furthermore, Project PTDC/EIA-EIA/102579/2008
is funded by FCT/MCE. C. Hendrickx was supported by the Fundação para a Ciência e a
Tecnologia (FCT) scholarship SFRH/BD/62979/2009 and the European Science
Foundation (ESF).
Author contributions
R.A., R.C., R.M., C.H., manuscript writing and figure preparation; R.A., R.C., thin-sections
and S.E.M.; R.M., R.C., F.B., synchrotron data acquisition and imaging; R.M., L.A.,
micro-PIXE analysis; R.M. and N.S., synchrotron radiation-based x-ray diffraction; C.H.,
R.A., anatomical description and phylogenetic analysis; R.C., cathodoluminescence; R.A.,
O.M., geology; R.A., R.C. and O.M., fossil preparation and excavation.
Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
License: This work is licensed under a Creative Commons
Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this
license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
How to cite this article: Araújo, R. et al. Filling the gaps of dinosaur eggshell phylogeny: Late
Jurassic Theropod clutch with embryos from Portugal. Sci. Rep. 3, 1924; DOI:10.1038/
srep01924 (2013).
8