Han Et Al. 2023 Qianlong Shouhu
Han Et Al. 2023 Qianlong Shouhu
net/publication/374585051
Exceptional Early Jurassic fossils with leathery eggs shed light on dinosaur
reproductive biology
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EARTH SCIENCES
© The Author(s) 2023. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. This is an Open Access article distributed under the terms of the Creative
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c d n
j
ma
sa pm
an de
10 cm
hy
e f
ri b 20 cm
Bulge
gr 2 cm
g ri i
ma
sa
de emf an
5 mm
2 cm
Figure 1. Skeletal morphology of Qianlong shouhu. Skeletal silhouettes of the (a) adult and (b) embryo showing preserved
bones (in gray) and standing postures. (c) Skull photograph and (d) line drawing in right lateral view, (e) maxillary teeth in
right lateral view and (f) right pes in posterior view of GZPM VN001 (adult). (g) Skull normal image and (h) transparency image
showing cheek teeth in left lateral view of GZPM VN004-2. (i) 3D reconstruction of the embryo GZPM VN006-1 showing the
prehatching posture, with skull elements in purple color; axial skeleton in green; scapula, forelimb and hindlimb in blue. an,
angular; de, dentary; emf, external mandibular fenestra; gr, groove; hy, hyoid bone; ma, maxilla; n, nasal; pm, premaxilla; ri,
ridge; sa, surangular.
003; Supplementary Fig. S2) and five clutches China and the Lower Jurassic Zhenzhuchong Mem-
of embryo-containing eggs (GZPM VN0 04-0 08; ber (possibly in Sinemurian), Ziliujing Formation
Fig. 2 and Supplementary Fig. S3). All fossils [10,11] (Supplementary Fig. S1a–c).
(GZPM VN0 01-0 08) are housed at the Guizhou
Provincial Museum (GZPM). Diagnosis
Qianlong differs from other sauropodomorphs in the
Locality and horizon following character states (autapomorphies marked
The locality and horizon are Zhuanpo, Pingba Dis- by *): a shallow concavity at the base of the pre-
trict, Anshun City, Guizhou Province, southwestern maxilla nasal process; relatively straight teeth with
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Figure 2. Egg clutch, eggs and eggshell microstructure of Qianlong shouhu. (a) Egg clutch GZPM VN005 preserving 16 eggs
and a fragmentary bone. (b) The embryonic-skeleton-containing egg GZPM VN006-1. (c) Close-up of eggshell of GZPM VN004-
1 showing cracked eggshell. (d and e) Radial thin sections and (h) line drawing of (d) showing the entire eggshell microstruc-
ture. (e, arrow) The eggshell covered by secondary calcite is thinner. (f) Radial thin section under polarized light and (g) Inverse
Pole figure map under EBSD analysis showing the mammillary cones with nucleation center (yellow arrows). (i) Tangential
thin section near the outer surface under PLM and (j) its line drawing showing interlocking eggshell units and elongated and
round pores (arrows). (k) Tangential thin section near the inner surface under TLM and (l) its line drawing showing isolated
eggshell units with nucleation center (arrows). cv, caudal vertebrae; es, eggshell; fe, femur; fi, fibula; Ii, left ilium ti, tibia.
labiolingually asymmetrical crowns and without end that is four times the mediolateral width of the
denticles; jaw articulation lower than dentary dor- distal end and with a small bulge on the lateral mar-
sal margin; a short retroarticular process; a very gin * (Fig. 1 and Supplementary Fig. S2).
small external mandibular fenestra; well-developed
nutritive foramina on the maxillary and dentary, the Description and comparisons
width of Metacarpal I being greater than its length; The skull and mandible (Fig. 1) share similar gen-
Metatarsal V with a strongly expanded proximal eral morphology to those of other early-diverging
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the large external naris is positioned anteriorly and Six embryos from two egg clutches display long
ventrally, and the dentary has a slightly down-turned bones through either direct exposure or CT imag-
anterior end and contributes to more than half of ing, and have a large medullary cavity and a very
the length of the mandible. There are also a number spongy cortex. Microstructures such as numerous
of derived cranial features, including a relatively primary cavities and abundant osteocyte lacunae
posteriorly positioned nasal with a short anteroven- (Supplementary Fig. S5) suggest fast growth [24].
tral process (also in Lufengosaurus [13], Mussaurus These embryos are probably in their late develop-
and other sauropodiforms [14]), a very small ex- mental stage as indicated by nearly full occupation
ternal mandibular fenestra (also in Yizhousaurus of the egg space by the skeleton (Fig. 1i). They dis-
[15], Riojasaurus [16] and many sauropodiforms), play a transitional prehatching posture between the
a high coronoid eminence and ventrally offset jaw crocodilians and living birds: the head is near the
articulation (also in Lufengosaurus, Jingshanosaurus pole and the hindlimbs are only partially crouched
[17], Yizhousaurus and most sauropodiforms), (Fig. 1i) as late-stage embryos of Massospondylus
relatively short surangular and angular (also in [25] and extant crocodilians [26,27], but the back
Yizhousaurus [15]), angular posteriorly posi- is curved along the pole and the hip is near the
tioned relative to the mandibular fenestra (also central portion of the egg as in those of early [28]
in Lufengosaurus and most sauropodiforms) and and living birds [29] as well as possibly ovirap-
labiolingually asymmetrical tooth crowns with- torids [30] and troodontids [31] (but see [27] for
out marginal denticles (also in Yunnanosaurus, a different interpretation of oviraptorid prehatch-
Irisosaurus and many sauropodiforms) and with sev- ing posture). All embryos are similar in the ossifica-
eral longitudinal ridges on the labial surface (also in tion degree and size (Fig. 3, Supplementary Fig. S6
Chuxiongsaurus [18]). and Supplementary Table S1), suggesting that Qian-
In the postcranial skeleton, morphologi- long has a synchronous hatching strategy and syn-
cal features shared with other early-diverging chronous breeding in this colonial nesting site.
sauropodomorphs (Supplementary Fig. S2) in- The embryos display several characteristics that
clude three sacral vertebrae; an elongated, laterally are shared with their adult counterparts (Fig. 1g–
arched scapula; a relatively short humerus with a i and Supplementary Fig. S6): the maxillary dor-
well-developed deltopectoral crest; a very stout sal process deflected distinctly from the anterior
Metatarsal I; a relatively small ilium with a short ramus at a large angle (∼70 degrees), the den-
pre-acetabular process and a long pubic pedun- tary proportionally long (∼60% of the mandibu-
cle; a long pubis with a large obturator foramen; lar length) and posteriorly bifurcated, the external
a long ischial shaft with a subtriangular cross sec- mandibular fenestra proportionally small, four pre-
tion; and a robust sigmoid femur longer than the maxillary teeth, relatively straight tooth crown, well-
tibia. Derived postcranial features include anterior developed nutritive foramina on the maxillary and
dorsals with a transversely expanded dorsal end dentary, the pubic apron long (>30% of the pu-
of the neural spine (also in Yizhousaurus [15] and bic length), the ilium with a short pre-acetabular
some other sauropodiforms), short anteriormost process and a relatively long postacetabular pro-
caudals (also in sauropods [12]), a short manus cess, a smooth convex dorsal margin, a long pu-
(also in Yizhousaurus [15], Jingshanosaurus [19] bic peduncle and the prominent plate-like femoral
and many other sauropodiforms [20]), robust fourth trochanter relatively proximally and medially
manual digits (also in Lufengosaurus [21] and some positioned.
sauropodiforms such as Yizhousaurus and Mussaurus However, the embryos also display some dif-
[22]), a relatively long pubic apron, Pedal Ungual ferences from the adults. Some of these differ-
I longer than all nonterminal phalanges (also in ences are ontogenetic variations also seen in other
Jingshanosaurus [19] and other sauropodiforms sauropodomorphs [14,32,33], including propor-
[23]) and a short Metatarsal V with a strongly tionally longer skull and mandible, a more vertical
expanded proximal end and a lateral bulge [un- anterior margin of the premaxilla and fewer teeth in
known in any other sauropodomorphs (Fig. 1f)] the embryos. Other differences, such as maxillary
(see Supplementary Data for more description and anterior ramus shallow and subtriangular in the em-
comparisons). Our phylogenetic analysis places bryos but deep in adults, the presence in the embryos
Qianlong as the sister taxon to Yunnanosaurus near but absence in the adults of a narrow ridge along
the base of Sauropodiformes (Supplementary the maxillary posterodorsal ramus (Fig. 1g) and
Fig. S4). retroarticular process long in the embryos but short
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a 1 1 2 3 4
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2
5 mm Posterior
2 mm
b 1 Lateral
2
3
c
1
2
3
Figure 3. Cross sections of the limb bones of Qianlong embryos derived from CT reconstruction. (a and b) GZPM VN006-2
showing that the forelimb is only slightly thinner than the hindlimb in Qianlong as indicated by the cross-section data. (a)
Humerus in anterior view. (b) Femur in posterior view. (c) GZPM VN004-2 femur in posterior view. GZPM VN004-2 is similar
in size to GZPM VN006-2 as indicated by the cross-section data.
in the adults, have not been reported previously in Nesting and eggs
the ontogenetic series of other sauropodomorphs
The five egg clutches containing the same type of
[14,32,33].
eggs are distributed in a small area of ∼15 m2 and the
Some proportional features (Supplementary
three adult/subadult skeletons are preserved with a
Table S1) indicate that the embryos have pro-
distance to the egg clutches ranging from 1 to 3 me-
portionally longer forelimbs and larger shoulder
ters. All fossils except GZPM VN002 (yellow surface
girdles than the adults—a phenomenon is also
color) are from purple silty mudstone and the latter
seen in Massospondylus and Mussaurus [34]. Limb
from a layer of purple siltstone ∼0.7 m above the for-
cross-section data display a similar pattern: humeral
mer fossil bed layer (Supplementary Figs S7–S11).
cross section is close in size to the femoral one in
The fossil-bearing beds are featured by massive
embryos (Fig. 3), but the difference is huge in adults.
fine brown mudstone, abundant calcium carbonate
Qianlong thus may have been quadrupedal at hatch-
nodules, along with slickensides and weak color
ing. Ontogenetic shifting from quadrupedalism to
mottling, indicating that they are paleosol origins
bipedalism has been proposed for early-diverging
and floodplain deposits of low energy (see more
sauropodomorphs based on data gathered in both
details in Supplementary Data). The general tapho-
limb proportions [25] or the body’s center of
nomical and sedimentary features are similar to
mass [35]. Our allometric growth analysis pro-
those of the fossil-bearing beds of several other
vides support for this proposal, and specifically
early-diverging sauropodomorphs [36,37] that
the humerus displays a negative allometry in the
have been suggested to possess such reproductive
growth of early-diverging sauropodomorphs but
behaviors as colonial nesting and site fidelity. The
near isometry or even a positive allometry in the
preserved Qianlong adult skeletons display a pros-
growth of sauropods (Fig. 6a). This suggests that
trating posture similar to that of some Plateosaurus
early-diverging sauropodomorphs are similar to
fossils that were interpreted as resulting from
sauropods in body plan at their early ontogenetic
miring [38].
stages, but differ in the growth pattern, which leads
The preserved egg clutches vary in size, with the
to the different body proportions at later ontogenetic
smallest clutch containing 3 eggs and the largest
stages.
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with 16 eggs (Fig. 2a and Supplementary Fig. S3), The presence of a calcareous layer is further
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and are much smaller in size than the largest known supported by our chemical analyses. Energy-
clutch of Massospondylus and Mussaurus containing dispersive spectroscopy (EDS) indicates that
34 eggs and 30 eggs, respectively [36,39], though Qianlong eggshell mainly consists of C, O and
the possibility of incomplete preservation leading Ca (Supplementary Table S12) and Raman spec-
to the small clutch sizes could not be dismissed. troscopy also detects the calcite signal from the
Most eggs are generally elliptical in outline. How- Qianlong eggshell (Fig. 4c and d). However, or-
ever, many small pits are observed on the outer sur- ganic matters are detected in the eggshells and the
face, leading to a somewhat irregular shape of the surrounding matrix and, interestingly, the Raman
eggs (Fig. 5c). Qianlong eggs have a diameter of spectra obtained from the Qianlong eggshell are
∼11.5 cm × 9.4 cm, which is more similar in size similar in calcite and organic matter signal patterns
to those of sauropods (ranging from 9 to 23 cm in to those from Mussaurus eggshell [3]. This suggests
egg diameter) [1,40] than to those of other early- that the signal pattern of organic matters revealed
diverging sauropodomorphs such as Massospondylus from Mussaurus eggshell [3] is not reliable evi-
and Mussaurus (∼6–7 cm in egg diameter) [25,41]. dence for soft-shelled eggs. The transmitted light
The eggs have a calcareous eggshell layer of microscopy (TLM), polarized light microscopy
160 ± 26 μm on average (n = 30, ranging from (PLM) and EBSD images confirm that Qianlong
115 to 230 μm, Fig. 2c–e). The irregular outer eggshell resembles other dinosaur eggshells at a
surface indicates eggshell weathering and thus the microstructural level, though the microstructure is
original Qianlong eggshell possibly is even thicker. less well preserved in Qianlong eggshell than in most
Qianlong thus has a calcareous eggshell that is Cretaceous dinosaur eggs that have been studied
considerably thicker than that of other known early- from this perspective.
diverging sauropodomorphs such as Massospondylus There are several lines of evidence supporting the
(80–100 μm) [2], much thicker than the calcare- identifications of the eggs of Qianlong and proba-
ous layer of all known soft-shelled eggs (usually bly other early-diverging sauropodomorphs as leath-
<60 μm) [42], but much thinner than that of most ery ones. First, their eggs have a shell thickness that
other non-avian dinosaur eggs (400–4750 μm) is similar to that of extant leathery eggs (usually
[43]. 70–200 μm) (see also Supplementary Table S8).
In radial thin sections, the eggshell consists of in- Second, Qianlong eggs display sharp edges of bro-
terlocking columnar eggshell units with a height-to- ken shells (Fig. 2c), as in some leathery eggs of ex-
wide ratio of ∼2 : 1 to 5 : 1 (Fig. 2d, e and h) and the tant turtles and hard-shelled eggs (Fig. 5b and c),
boundaries between the interlocking eggshell units and they further resemble leathery eggs in having
are irregular (Fig. 2f and g). Round and elongated small eggshell pieces when eggs are broken (Fig.
pores occasionally appear between adjacent eggshell 5b). Finally, our statistic analyses of relative eggshell
units (Fig. 2i and j). Quantitative analysis indicates thickness (Fig. 5f) and the relative size of eggshell
that Qianlong had relatively high eggshell porosity pieces (Fig. 5g) demonstrate that Qianlong eggs are
(Supplementary Table S5) and, by combining the more similar to leathery eggs than to either hard-
egg mass data, our analysis indicates that Qianlong shelled eggs or soft-shelled eggs. In summary, the
had covered nests (Supplementary Fig. S13) as in relatively thin eggshell thickness compared with the
most non-pennaraptoran archosaurs [44]. Towards egg mass, the rugose egg surface, the slightly irreg-
the inner surface, the eggshell units become isolated ular egg shape and the strongly pieced eggshells
from each other (Fig. 2k and l). At the inner sur- provide strong support for the leathery nature of
face of the eggshell, the mammillary cone exhibits eggs of Qianlong and probably other early-diverging
a radial arrangement of calcite grains and a small sauropodomorphs (Fig. 5 and Supplementary Fig.
rounded nucleation center (Figs 2d–h, k, l and 4a, S3c).
c), as in turtles and all other dinosaurs including
birds [45] (Supplementary Figs S14 and S15). Elec-
tron backscatter diffraction (EBSD) imaging shows
that the mammillary cones continue by large vertical
Evolution of selected reproduction
prism-shaped calcite grains in the outer portion of features
the eggshell (Fig. 2g) as in typical dinosaur eggshells To better understand the evolution of avian repro-
(e.g. Placoolithus, Supplementary Fig. S14f); scan- ductive biology, we performed ancestral-state recon-
ning electron microscope (SEM) imaging reveals struction (ASR) analyses to trace the evolution of
that there are numerous tiny vesicles in the calcite egg size and shape as well as eggshell type, mi-
crystals (Fig. 4b), resembling those of Cretaceous di- crostructure and thickness. The new data sets were
nosaur eggshells [46,47]. compiled from several recent studies [2,3,6,48] but
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Figure 4. Microstructure and Raman spectra of Qianlong eggshell. (a) Radial section under SEM showing the nucleation
centers of two eggshell units (arrows). (b) Radial section under SEM showing numerous cavities (white arrows) and tiny
vesicles (yellow arrows) throughout the whole eggshell. (c) Radial thin section of the eggshell under normal light showing
eggshells (es) and nucleation centers (yellow arrows). (d) Raman point spectra were acquired at the positions labeled with
the red dots in (c): 1. Epoxy resin; 2. Secondary calcite on the outer surface of the eggshell; 3, 4. Eggshell; 5. Organic matters
in sediments; 6. Calcite in sediments.
with significant expansion, and they contain 210 di- also been detected in some lepidosaur lineages and
apsid taxa with both ascertained systematic posi- in the evolution of sauropodomorphs and neog-
tions and relevant reproduction data for our analyses naths, leading to some of the smallest eggs found
(Fig. 6 and Supplementary Figs S16 and S17). in some sauropodomorph clades, among the known
Our egg-size ASR analyses show that the evo- archosaurian clades.
lution of relative egg size (egg volume relative to Egg-shape evolution displays a different pattern
adult body mass) displays a decreasing trend from from size evolution. Egg shape (measured by using
the base of the Diapsida to that of the Saurischia, the elongation index) is generally conservative
followed by an egg-size-increase trend from early along the line to living birds in diapsid evolution:
theropods to the crown bird node (Fig. 6c and nearly all nodes (e.g. the Diapsida, Archelosauria,
Supplementary Figs S18 and S19). The former Archosauria, Ornithodira and Aves) except sev-
trend leads to plesiomorphically smaller eggs in Di- eral non-avialan dinosaurian nodes display an
nosauria (with the exception of turtles) and the lat- egg elongation index of 0.13–0.15 (Fig. 6d and
ter to plesiomorphically larger eggs in Aves com- Supplementary Figs S20 and S21). This lack of
pared with all other diapsid groups, though the shape change is also seen in most crown bird clades,
most significant egg-size increase occurred early in in stark contrast to most reptilian groups and their
theropod evolution. Meanwhile, an egg-size-increase subclades that display either a much smaller or
trend is also seen in some lineages of lepidosaurs, tur- a much larger egg elongation index (Fig. 6d and
tles, crocodilians, pterosaurs, ornithischians, ovirap- Supplementary Figs S20 and S21). The former is
torosaurians, palaeognaths and neognaths, though seen in sauropodomorphs, ornithischians, turtles
only the trend in the oviraptorosaurian and pa- and a few lepidosaur clades, which have nearly
leognath evolution has been relatively well sup- rounded eggs, and the latter is present in non-avialan
ported by the data. An egg-size-decrease trend has theropods, pterosaurs, crocodilians and some
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a b c d e
1 cm 1 cm 2 cm 1 cm
2 mm 1 mm 2 mm 1 cm
2 cm
f 4.0 g 5.0
LOG eggshell thickness (μm)
2.0 3.5
Figure 5. Photographs and scaling of diapsid eggshell type, thickness and fragment size. (a) Soft eggshell. Pantherophis guttatus, strongly folded
eggshell without broken fragments. (b and c) Leathery eggshells. (b) Pseudemys nelson, moderately folded eggshell with small fragments (∼1–2 mm).
(c) Qianlong shouhu, showing rugose eggshell surface with small fragments (∼2 mm). (d and e) Rigid eggshells. (d) Gallus gallus domesticus, showing
large eggshell fragments. (e) Elongatoolithus magnus (CUGW EH023), showing large eggshell fragments. (f) Plot of LOG egg mass vs. LOG eggshell
thickness in diapsids. (g) Plot of LOG egg volume vs. LOG eggshell fragment sizes. Both charts support that Qianlong probably laid leathery eggs.
lepidosaur clades, which show much more elon- seen in sauropodomorphs, ornithischians, some lin-
gated eggs. The theropod egg elongation leads to eages of oviraptorosaurian theropods, crocodilians,
the most elongated diapsid eggs in oviraptorosaurs, turtles and lepidosaurs.
but would later be reversed to the plesiomorphic, Although the homologous relationships of diap-
slightly elongated eggs that are inherited by all crown sid eggshells are highly debated [48,49], the eggshell
bird clades. units are widely accepted to be the basic components
Similarly, the relative eggshell thickness (eggshell of the calcareous shell layer [50]. Thus, the eggshell
thickness relative to egg volume) also displays a rel- unit evolution is key to our understanding of diapsid
atively complex evolutionary pattern (Fig. 6e and egg evolution. Our ASR analyses of the eggshell-
Supplementary Figs S22 and S23). Along the line to unit-elongation index (EI, the ratio of eggshell unit
extant birds in archosaur evolution, there is an evo- length to width) show that there is an evolutionary
lutionary trend of eggshell-thickness decrease from trend of eggshell unit elongation from the base of
the base of the group to that of the Saurischia, fol- Archelosauria to that of Pennaraptora, and along
lowed by a significant eggshell-thickness increase some lineages of neognath and paleognath birds,
early in theropod evolution. An evolutionary trend of oviraptorosaurian theropods, sauropodomorphs
eggshell-thickness decrease is also seen in neognaths, and turtles. Meanwhile, an opposite trend is present
paleognaths, enantiornithines, some turtle lineages in enantiornithines and some paleognath, neog-
and some lepidosaur lineages whereas the reverse is nath, turtle, crocodilian and sauropodomorph
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a 3.00 b Enantiornithes
2.50
Neognathae Sauropodomorpha
LOG humerus length (cm)
Squamata
0.50 1.00 1.50 2.00 2.50 3.00 Ornithischia
LOG femur length (cm)
Palaeognathae
c Soft-shelled egg
Leathery-shelled egg
300 250 200 150 100 50 0 Ma Testudines Hard-shelled egg
Lepidosauria
d
Testudines 300 250 200 150 100 50 0 Ma
Lepidosauria
Crocodilia
Archelosauria
Testudines
Archosauria Pterosauria
Figure 6. Sauropodomorph growth strategies and diapsid reproduction evolution. (a) Regression analysis shows growth trajectories of selected
sauropodomorphs. (b) Eggshell type ASR under hierarchal Bayes framework with new scoring and ARD model (two rate classes; using majority rule
consensus tree of run1 in the first dating analysis). A simplified time-calibrated diapsid tree showing the (c) egg-size evolution, (d) egg elongation index,
(e) eggshell-thickness evolution and (f) eggshell unit based on our ASR analyses (Supplementary Figs S18–S25). Color changes from blue to red indicate
an increase in all values. Arche, Archelosauria; Archo, Archosauria; Avem, Avemetatarsalia; Diap, Diapsida; Thero, Theropoda.
lineages (Fig. 6f and Supplementary Figs S24 and ones [45,50–52] (see Supplementary Data), though
S25). Among diapsids, some oviraptorosaurian and this classification oversimplifies the great variety
troodontid clades have the most elongated eggshell of eggshell morphologies [48]. Nevertheless, a
units while some crocodilian and turtle clades have calcareous layer formed by eggshell units character-
the shortest ones. izes both leathery and hard-shelled eggs, the appear-
Extant amniotic eggs are traditionally classi- ance of which represents a key event in egg evolu-
fied into soft-shelled, leathery and hard-shelled tion [2]. Eggs of early-diverging sauropodomorphs
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are only known in three species, but are contro- eggshell units, and probably leathery. Along the line
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versial in their morphologies. The calcareous layer to living birds in dinosaur evolution, the most sig-
of Massospondylus eggshells seems to be composed nificant change in egg morphology occurred early in
of columnar structural units, but whether they are theropod evolution and stem birds closely resemble
original eggshell units is uncertain due to severe non-avialan theropods and particularly non-avialan
recrystallization of the eggshells [2]; the known maniraptorans in egg morphology. Except for rel-
Lufengosaurus eggshell is composed of crown-shaped atively large egg size, extant birds either inherited
eggshell units with radially arranged calcite crystals, their theropod ancestral condition (e.g. relatively
comparable to the inner portion of the Qianlong thick eggshell and long eggshell units) or reversed
eggshell (Fig. 2e–h), suggesting that the preserved to the primitive condition (e.g. relatively short eggs)
Lufengosaurus eggshells likely represent eggshell in- in egg morphology. The discovery of Qianlong and
terior with the exterior being weathered away; the our analyses clearly show that the evolution of the
soft-shelled nature of Mussaurus eggs has been in- dinosaur reproduction system is a complex process
ferred based on the chemical composition revealed and the evolution of some important reproduction
by Raman spectra [3], but our comparative chem- features such as egg size and shape and eggshell
ical data from Qianlong eggs support the argument thickness are more likely to have been driven by
that the chemical evidence for the presence of soft- multiple factors rather than by a single factor such as
shelled eggs in Mussaurus needs be re-evaluated phylogeny, development or environment.
[51]. Our eggshell type ASR analyses incorporate
new data from Qianlong and other key taxa and
are conducted with consideration of temporal and CONCLUSION
character scoring uncertainty—issues that might This study reports some exceptional fossils of a new
have affected significantly the ASR analyses [48]. early-diverging sauropodomorph dinosaur, Qianlong
For example, to consider temporal and character shouhu gen. et sp. nov., from the Lower Juras-
scoring uncertainty in eggshell type ASR, we re- sic Ziliujing Formation of southwestern China and
spectively used 22 different time-scaled trees and makes several novel findings pertaining to diap-
two different criteria for identifying eggshell types sid reproduction biology: (i) The early-diverging
(Supplementary Data and https://figshare.com/s/ sauropodomorph Qianlong has relatively large eggs
14374b47d33d96aef963). These analyses produced with a relatively thick calcareous shell formed
similar and robust results, and recovered pterosaurs by prominent mammillary cones compared with
as ancestrally soft-shelled and Archelosauria, Tes- other early-diverging sauropodomorphs, a transi-
tudines, Archosauria, Avemetatarsalia, Dinosauria tional prehatching posture between the crocodil-
and Saurischia as ancestrally leathery eggshell in ians and living birds, and a synchronous hatch-
most results (Fig. 6b and Supplementary Figs S26– ing pattern. (ii) Qianlong and other early-diverging
S32). sauropodomorphs have leathery eggs. (iii) ASR anal-
Some results of our analyses are different from yses demonstrate that the first dinosaur eggs were
those of some previous studies [4,8,48]. For exam- probably leathery, elliptical and relatively small, but
ple, the first dinosaur eggs were suggested to be either with relatively long eggshell units, and that egg shape
hard [52] or soft [3]; other studies suggest that the is generally conservative among extant birds and the
major changes in the avian reproduction system have most significant change in reptilian egg morphology
occurred incrementally, including an evolutionary occurred early in theropod evolution. These findings
trend of increasing egg size along the line to crown are significant to our knowledge of the reproductive
birds [4,8] and an increasing eggshell thickness after biology of diapsids, particularly of dinosaurs.
the Early Jurassic corresponding to an increase in
global atmospheric oxygen during the same tempo-
ral period [2]. However, our study provides strong METHODS
evidence for the leathery eggs in early-diverging
sauropodomorphs and suggests a leathery eggshell Phylogenetic analyses
origin for major diapsid subclades including the Di- We analyse a recently published data set for
nosauria; our study also reveals a complex evolution- sauropodomorph phylogeny [36] with Qianlong
ary history of egg size and eggshell thickness along added in Supplementary Table S3. A total of 80 taxa
the line to crown group birds. Most significantly, and 419 characters were included in the data matrix.
our analyses indicate that dinosaurs ancestrally had The analysis was run using TNT V. 1.5 [53] with the
distinct eggs compared with other reptilian groups, maximum trees set to 10 0 0 0. All the characters were
which were relatively small, moderately elongated equally weighted and 41 additive characters were set
and thin-shelled but with moderately elongated [36]. A heuristic search using a new technologies
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Natl Sci Rev, 2024, Vol. 11, nwad258
algorithm was used, with 100 hits to minimum tion [48]. We assembled an informal supertree man-
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length, followed by tree swapping using TBR (tree ually in Mesquite v3.6.1 and used hidden Markov
bisection reconnection) on the trees in memory (see chain model that considers rate heterogeneity and
details in Supplementary Data). performed ASR analyses of eggshell type under a Hi-
erarchal Bayesian framework in RevBayes.1.1.1 using
Computed tomographic scan all rate different model (ARD) under two hidden rate
classes. Relative egg size and relative eggshell thick-
and 3D reconstruction
ness were determined by using phylogenetic linear
Four embryo-containing eggs were scanned using regression with Log10 transformed data. Residu-
a Phoenix Vtomex M micro-computed tomogra- als taken from the regression models were used to
phy scanner at the Yinghua Inspection and Test- indicate the relative egg size and relative eggshell
ing Shanghai Company and the Key Laboratory of thickness. The phylogenetic linear regression analy-
Vertebrate Evolution and Human Origin of Chinese ses were performed in R 4.1.3 with the package ‘ca-
Academy of Sciences, IVPP. Scanning parameters per’ and Pagel’s Lambda was used to consider the
were set to a tube voltage of 180–200 KV and a phylogenetic signal (Supplementary Fig. S17). Iden-
current of 100–150 μA with a voxel size of 22.49– tically, we performed ASR on log 10 transformed
35.044 μm3 (Supplementary Table S13). Recon- egg EI (ratio of egg long axis to short axis) and
struction of radiographs was performed using the eggshell unit index (ratio of eggshell unit depth to
software Mimics 17 at the IVPP. width) with supertrees rescaled by Pagel’s Lambda
by using the function ‘fastAnc’ (see detailed in
Raman analyses Supplementary Data).
In situ Raman microspectroscopy was conducted us-
ing a WITec α300 Confocal Raman system coupled SUPPLEMENTARY DATA
with a Peltier cooled EMCCD detector at the State Supplementary data are available at NSR online.
Key Laboratory of Biogeology and Environmental
Geology, China University of Geosciences (Wuhan).
Laser excitation was provided at 532 nm with 7.9 mW
ACKNOWLEDGEMENTS
of output laser power at the surface of the sample. We thank Guizhou Provincial Museum for permission and re-
Each sample was scanned in the spectral range from search support; Krishna Hu for editing the manuscript; Shihui
0 to 40 0 0 cm−1 . The integration time for each spec- Mei, Zhenhuang Luo and Song Luo for collecting the specimens;
Jinchao Ding for preparing the specimens; Shunyi Shi for Fig. 1b;
trum was 3 s and the number of accumulations was
Minghui Ren for Fig. 1a and b; Honglang Zhang for Fig. 2h, j and
10. Software WITec Project Five 5.1 Plus was used to l; Chang Xu and Seung Choi for helping on EBSD analysis; Kenan
process the Raman spectra. Cao for helping on Raman analysis; Xun Jin for helping on SEM
and EDS analyses; Yemao Hou for helping on CT scanning; Xian-
Allometric growth analysis duo Dai and Yuzheng Ke for helping on sedimentological analysis;
Jichao Wang and Li Jian for providing sea turtle eggshells; David
Allometric growths of three non-sauropod J. Varricchio, Jason R. Moore, Haijun Song, Wenchao Yu, Jiang-
sauropodomorph species and five sauropod species hai Yang, Haishui Jiang and Xulong Lai for their useful discussions
were investigated by using a bivariate plot of the and suggestions.
humeral length relative to the femoral length of 33
individuals representing different ontogenetic stages
of these eight species (Supplementary Table S4).
FUNDING
Unitary linear regression analyses were performed This work was supported by the National Natural Science Foun-
to detect the allometric relationships between dation of China (42288201, 42372036, 41688103 and 41972021).
the log-transformed humerus and femur in Excel
(2016). AUTHOR CONTRIBUTIONS
X .X ., F.L.H. and Y.L.Y. designed the project and experiments.
ASR analyses F.L.H. and S.K.Z. prepared all the drawings. S.K.Z. and R.W.
prepared thin sections of eggshells and bones. F.L.H., Y.L.Y. and
The sampled taxa cover major reptilian clades, in-
R.W. collected the data for the ancestral-state reconstruction, did
cluding crocodilians, birds, non-avian dinosaurs, CT Scanning, Raman and EBSD analyses. Y.L.Y. performed all
pterosaurs, turtles, lepidosaurs and choristoderes the ancestral-state reconstruction. Y.F.W. provided all the pho-
(Supplementary Tables S9 and S10) and in total the tographs in Supplementary Fig. S7. R.Z., Y.F.W., H.Y.C., S.F.C
data sets include 210 taxa. Here we use two criteria and C.L. contributed material and material information. X.J.W.,
(new scoring and ratio scoring) to do ASR analy- H.Y.C., T.Z.W., Y.F.W. and F.L.H. contributed to field work. F.L.H.
sis for they are widely used in eggshell type defini- did the phylogenetic analysis and allometric growth analyses. Q.Z.
Page 11 of 13
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contributed to bone histological analysis. X .X ., F.L.H., Y.L.Y. and 15. Zhang Q-N, You H-L and Wang T et al. A new sauropodiform
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S.K.Z. wrote the manuscript. dinosaur with a ‘sauropodan’ skull from the Lower Jurassic
Lufeng Formation of Yunnan Province, China. Sci Rep 2018; 8:
Conflict of interest statement. None declared. 13464.
16. Barrett PM, Upchurch P and Zhou XD et al. The skull of Yun-
nanosaurus huangi Young, 1942 (Dinosauria: Prosauropoda)
from the Lower Lufeng Formation (Lower Jurassic) of Yunnan,
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© The Author(s) 2023. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. This is an Open Access article distributed under the terms of the Creative
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