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Abstract and Figures

Calcified eggshells protect developing embryos against environmental stress and contribute to reproductive success¹. As modern crocodilians and birds lay hard-shelled eggs, this eggshell type has been inferred for non-avian dinosaurs. Known dinosaur eggshells are characterized by an innermost membrane, an overlying protein matrix containing calcite, and an outermost waxy cuticle2,3,4,5,6,7. The calcitic eggshell consists of one or more ultrastructural layers that differ markedly among the three major dinosaur clades, as do the configurations of respiratory pores. So far, only hadrosaurid, a few sauropodomorph and tetanuran eggshells have been discovered; the paucity of the fossil record and the lack of intermediate eggshell types challenge efforts to homologize eggshell structures across all dinosaurs8,9,10,11,12,13,14,15,16,17,18. Here we present mineralogical, organochemical and ultrastructural evidence for an originally non-biomineralized, soft-shelled nature of exceptionally preserved ornithischian Protoceratops and basal sauropodomorph Mussaurus eggs. Statistical evaluation of in situ Raman spectra obtained for a representative set of hard- and soft-shelled, fossil and extant diapsid eggshells clusters the originally organic but secondarily phosphatized Protoceratops and the organic Mussaurus eggshells with soft, non-biomineralized eggshells. Histology corroborates the organic composition of these soft-shelled dinosaur eggs, revealing a stratified arrangement resembling turtle soft eggshell. Through an ancestral-state reconstruction of composition and ultrastructure, we compare eggshells from Protoceratops and Mussaurus with those from other diapsids, revealing that the first dinosaur egg was soft-shelled. The calcified, hard-shelled dinosaur egg evolved independently at least three times throughout the Mesozoic era, explaining the bias towards eggshells of derived dinosaurs in the fossil record.
Hierarchical cluster analysis of biomineralization signatures preserved in eggshell proteins (extant samples) and their fossilization products (fossil samples) The topology represents a cluster analysis of n = 24 selected eggshell protein and PFP bands (Methods). Sampling of both biomineralized proteins (in situ analysis) from hard-shelled eggs and extracted, non-biomineralized membranes from soft and decalcified hard-shelled (Caiman, Alligator, Emys, Mesoclemmys, Phrynops and Gallus) eggs avoids phylogenetic attraction of the included fossil samples, and thereby allows eggshell clustering on the basis of the protein and PFP biomineralization signal. Two separate clusters of biomineralized and non-biomineralized eggshell proteins/PFPs are recovered. Pink nodes illustrate biomineralized egg proteins/PFPs, and blue nodes represent non-biomineralized eggshell proteins/PFPs. The egg icons illustrate whether samples represent originally hard or soft eggshell. One spectrum only was used for Mussaurus, as there is not much compositional variation across the eggshell (Fig. 1e), whereas all three eggshell spectra were sampled for Protoceratops, owing to the differences in composition across the egg section (Fig. 1d). Hard-shelled Alligator and turtle eggshells were excluded from this biomineralization analysis, as they do not produce any substantial organic signal with the spectroscopy protocol used (Supplementary Information and ref. ⁴⁴). Both Protoceratops and Mussaurus eggshells are nested within the cluster of originally non-biomineralized eggshell proteins/PFPs.
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406 | Nature | Vol 583 | 16 July 2020
Article
The first dinosaur egg was soft
Mark A. Norell1 ✉, Jasmina Wiemann2 ✉, Matteo Fabbri2 ✉, Congyu Yu1, Claudia A. Marsicano3,
Anita Moore-Nall4, David J. Varricchio4, Diego Pol5 & Darla K. Zelenitsky6
Calcied eggshells protect developing embryos against environmental stress and
contribute to reproductive success1. As modern crocodilians and birds lay
hard-shelled eggs, this eggshell type has been inferred for non-avian dinosaurs.
Known dinosaur eggshells are characterized by an innermost membrane, an overlying
protein matrix containing calcite, and an outermost waxy cuticle2–7. The calcitic
eggshell consists of one or more ultrastructural layers that dier markedly among the
three major dinosaur clades, as do the congurations of respiratory pores. So far, only
hadrosaurid, a few sauropodomorph and tetanuran eggshells have been discovered;
the paucity of the fossil record and the lack of intermediate eggshell types challenge
eorts to homologize eggshell structures across all dinosaurs8–18. Here we present
mineralogical, organochemical and ultrastructural evidence for an originally
non-biomineralized, soft-shelled nature of exceptionally preserved ornithischian
Protoceratops and basal sauropodomorph Mussaurus eggs. Statistical evaluation of
insitu Raman spectra obtained for a representative set of hard- and soft-shelled, fossil
and extant diapsid eggshells clusters the originally organic but secondarily
phosphatized Protoceratops and the organic Mussaurus eggshells with soft,
non-biomineralized eggshells. Histology corroborates the organic composition of
these soft-shelled dinosaur eggs, revealing a stratied arrangement resembling turtle
soft eggshell. Through an ancestral-state reconstruction of composition and
ultrastructure, we compare eggshells from Protoceratops and Mussaurus with those
from other diapsids, revealing that the rst dinosaur egg was soft-shelled. The
calcied, hard-shelled dinosaur egg evolved independently at least three times
throughout the Mesozoic era, explaining the bias towards eggshells of derived
dinosaurs in the fossil record.
Hard-shelled eggs are an important character defining modern birds
and are thought to have had a key role in their survival through the
Cretaceous–Palaeogene extinction (approximately 66 million years
ago)
1
. The calcified avian eggshell stands in contrast to the primitive
amniote eggshell condition: early amniotes and more primitive tet-
rapods
2–7
laid soft eggshells. Extant archosaurs share assembly-line
oviducts
8
, corpus luteum morphology
9
and the embryonic resorption
of eggshell calcite—factors that would seem to suggest homology of
hard, calcitic eggshell among crocodilians and all dinosaurs, non-avian
and avian
1014
. However, pterosaurs—the sister group to dinosauro-
morphs—laid soft eggs15–18.
Non-avian dinosaurs are thought to have shared with crocodil-
ians, extant birds and most turtles an innermost shell membrane6, a
biomineralized protein matrix and an outer cuticle6. Such architecture
is found in most previously described dinosaur eggs, regardless of
shape, size or colour
19
. Both the shell membrane and the biomineral-
ized protein matrix are arranged in multiple layers of varying internal
patterning. Calcitic dinosaur eggs
20
are generally considered hard
tissues, and their fossil record is patchy in terms of diversity and age21.
Only eggs of afewtaxa—such as ornithopods, sauropodomorphs,
titanosaurs and tetanurans—have been reliably identified
2126
. The
vast majority of these eggs arefrom the Cretaceousperiod
2128
. How-
ever, the diversity of dinosaur taxa from the Triassicperiod to the
Cretaceous suggests that the apparent biases in the egg fossil record
cannot be explained solely by preferential preservation of certain
nesting sites, as previously hypothesized. Even in highly fossiliferous
localities, such as the Mongolian Djadoktha2629 and the Tugrugeen
Shireh site30, where eggs and embryonic remains are relatively com-
mon, eggshells attributable to more basal dinosaur taxa have not
been recovered.
Previous attempts to homologize archosaur eggshell ultrastructures
failed24,29 because of fundamental differences in the layer organiza-
tion
2329,31
. Ornithopod eggshells
2325,29
have one calcified spherulitic
layer. Basal sauropodomorph eggshell32–35 consists primarily of a
thick membrane covered by a thin, nondescript calcitic layer3235, and
titanosaurid sauropod eggshells
22,36
possess a single, well-calcified
spherulitic layer on a thinner membrane22,36,37. The number of calcified
ultrastructural layers in theropod eggshells varies between one and
three22–24,2629,31,38,39. Current hypotheses assume a single evolutionary
origin of the dinosaurian calcified egg11–13.
https://doi.org/10.1038/s41586-020-2412-8
Received: 19 March 2019
Accepted: 14 May 2020
Published online: 17 June 2020
Check for updates
1Division of Vertebrate Paleontology, American Museum of Natural History, New York, NY, USA. 2Department of Geology & Geophysics, Yale University, New Haven, CT, USA. 3Departamento de
Ciencias Geológicas, Universidad de Buenos Aires, Buenos Aires, Argentina. 4Department of Earth Sciences, Montana State University, Bozeman, MT, USA. 5CONICET, Museo Paleontológico
Egidio Feruglio, Trelew, Argentina. 6Department of Geoscience, University of Calgary, Calgary, Alberta, Canada. e-mail: norell@amnh.org; jasmina.wiemann@yale.edu; matteo.fabbri@yale.edu
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... Soft-shelled eggs are mostly proteinaceous and extremely rare in the fossil record (Legendre, Rubilar-Rogers, Musser, et al., 2020;Norell et al., 2020;Stewart, 1997); almost all fossil eggshells consist only in a CL with shell units (Mikhailov, 1997b). Eggshell parataxonomy, primarily developed to identify isolated fossil eggs of unknown egg layer, is based on the morphology of these shell units and cannot be used to describe eggs that lack them (Packard & DeMarco, 1991;Schleich & Kästle, 1988). ...
... eggshell thickness, porosity, calcium content, water vapor conductance) with life history traits in birds (Attard & Portugal, 2021;Birchard & Deeming, 2009, 2015McClelland et al., 2021;Portugal et al., 2014), dinosaurs including birds (Legendre & Clarke, 2021;Tanaka et al., 2015), archosaurs (Tanaka & Zelenitsky, 2014), squamates (Hallmann & Griebeler, 2015), non-avian reptiles (D'Alba et al., 2021), or across amniotes, albeit with relatively small samples (Legendre, Rubilar-Rogers, Musser, et al., 2020;Stein et al., 2019). Some of these studies have also reconstructed ancestral states for these traits -discretized in some cases -and identified correlates that influenced these evolutionary patterns (Attard & Portugal, 2021;D'Alba et al., 2021;Legendre & Clarke, 2021;Legendre, Rubilar-Rogers, Musser, et al., 2020;McClelland et al., 2021;Norell et al., 2020;Portugal et al., 2014;Stein et al., 2019;Tanaka et al., 2015). These studies have initiated what is perhaps the most interesting and controversial debate in this new field of quantitative eggshell research (Lindgren & Kear, 2020): what was the structure of the ancestral eggshell in dinosaurs, archosaurs, and reptiles as a whole? ...
... Recent descriptions of exceptionally preserved fossil softshelled eggs assigned to early diapsids (Hou et al., 2010), pterosaurs (Grellet-Tinner et al., 2014;Unwin & Deeming, 2008;Wang et al., 2014Wang et al., , 2017, non-avian dinosaurs (Norell et al., 2020;Stein et al., 2019) and, possibly, marine reptiles (Legendre, Rubilar-Rogers, Musser, et al., 2020), have led studies to investigate via ancestral state reconstructions (ASR) the binary trait "soft-shelled/hardshelled" (sometimes with a third intermediate state, referred to as a semi-rigid eggshell) on the phylogenetic tree of Reptilia. One study recovered the first dinosaur and archosaur egg as soft-shelled (Norell et al., 2020) and another with both as hard-shelled (Legendre, Rubilar-Rogers, Musser, et al., 2020), which was until then the scientific consensus. ...
Article
Reptile eggshell ensures water and gas exchange during incubation and plays a key role in reproductive success. The diversity of reptilian incubation and life history strategies has led to many clade-specific structural adaptations of their eggshell, which have been studied in extant taxa (i.e. birds, crocodilians, turtles, and lepidosaurs). Most studies on non-avian eggshells were performed over 30 years ago and categorized reptile eggshells into two main types: “hard” and “soft” – sometimes with a third intermediate category, “semi-rigid.” In recent years, however, debate over the evolution of eggshell structure of major reptile clades has revealed how definitions of hard and soft eggshells influence inferred deep-time evolutionary patterns. Here, we review the diversity of extant and fossil eggshell with a focus on major reptile clades, and the criteria that have been used to define hard, soft, and semi-rigid eggshells. We show that all scoring approaches that retain these categories discretize continuous quantitative traits (e.g. eggshell thickness) and do not consider independent variation of other functionally important microstructural traits (e.g. degree of calcification, shell unit inner structure). We demonstrate the effect of three published approaches to discretizing eggshell type into hard, semi-rigid, and soft on ancestral state reconstructions using 200+ species representing all major extant and extinct reptile clades. These approaches result in different ancestral states for all major clades including Archosauria and Dinosauria, despite a difference in scoring for only 1–4% of the sample. Proposed scenarios of reptile eggshell evolution are highly conditioned by sampling, tree calibration, and lack of congruence between definitions of eggshell type. We conclude that the traditional “soft/hard/semi-rigid” classification of reptilian eggshells should be abandoned and provide guidelines for future descriptions focusing on specific functionally relevant characteristics (e.g. inner structures of shell units, pores, and membrane elements), analyses of these traits in a phylogenetic context, and sampling of previously undescribed taxa, including fossil eggs.
... Most researchers focus on hard-shelled eggs (such as dinosaur eggs), while there are few studies on softshell specimens. Mainly because the soft eggs have poor preservation potential, and only a small number of cases have been reported [34][35][36]. To obtain more information from these valuable and rare soft egg specimens, researchers have shown an increased interest in element and chemical analysis [37], especially in using Raman spectroscopy for its non-destruction. ...
... To obtain more information from these valuable and rare soft egg specimens, researchers have shown an increased interest in element and chemical analysis [37], especially in using Raman spectroscopy for its non-destruction. Recently, Raman spectroscopy has played an important role in soft egg studies, such as a giant egg from the Late Cretaceous of Antarctica (Antarcticoolithus) [35], the ornithischian Protocertops, and the basal sauropodomorph Mussaurus eggs [36]. ...
... If fluorapatite is the result of diagenesis, it could therefore conceivably be hypothesized that H. tianshanensis may lay soft eggs. Soft eggs, such as secondarily phosphatized protocertops [36] and Antarcticoolithus bradyi [35], both contain calcium phosphate. In fact, diagenetic alteration of the mineral composition of membrane testacea to apatite is relatively common in fossil eggs [27,65,66]. ...
Article
Full-text available
Pterosaur eggs can offer information about pterosaur reproductive strategies and are extremely precious because only a small number of specimens have been discovered. Previous studies have mainly focused on morphological descriptions of pterosaur eggs and their embryos while the chemical composition of pterosaur eggs has received little attention. The conventional view believed that the eggshell was composed of calcite. However, previous SEM–EDS results for Hamipterus tianshanensis showed that the eggshell contains phosphorus. Therefore, the object of this research is to determine the mineral composition of the eggshell of H. tianshanensis. Two eggs were analyzed by scanning electron microscopy coupled with energy dispersive X-ray spectrometry (SEM–EDS) and Raman spectroscopy. The SEM–EDS results show that both surface and cross section are porous and characterized by small irregularly shaped particulates. Moreover, the distribution of Ca and P has a strict coincidence in the cross-section of eggshells. Furthermore, neither the intense peaks of calcite nor organic peaks can be observed by Raman spectroscopy in eggshells. Meanwhile, the Raman spectroscopy mapping analysis result shows a sharp and intense peak at approximately 966 cm−1 among the white eggshell, which can be hard evidence that H. tianshanensis eggs are mainly composed of calcium phosphate. Combined with the present of F in the eggshell, it can be inferred that fluorapatite Ca5(PO4)3F is the main mineral. The fluorapatite eggshell can be interpreted in two ways. One explanation is that H. tianshanensis laid apatite-shelled eggs, similar to living Salvator merianae, and the bioapatite transformed to fluorapatite over geological time. Another possible explanation is that the fluorapatite comes from the result of phosphatization of soft egg membrane tissues through taphonomic processes, indicating that H. tianshanensis might have laid soft eggs. Regardless, the results show that fluorapatite, rather than calcite is the main preserved mineral composition of H. tianshanensis eggshell, correcting the previous view. This study contributes to the present understanding of the mineral composition of pterosaur eggshells and may offer some insight into the pterosaur reproduction pattern.
... In addition, the eggs of the early branching sauropodomorph Mussaurus patagonicus have been recently redescribed as soft-shelled instead of mineralized (Norell et al. 2020) although this claim has been disputed (Alleon et al. 2021; but see Wiemann and Briggs 2021 for a reply). We describe each of these oofamilies' records from South America below. ...
... Since the original works of Mones (1980), these eggs have been attributed to sauropods, and lately to neosauropods or even titanosaurs based on (1) large egg size; (2) rounded shape; (3) clutch arrangement; and (4) presence, sometimes exclusively, of titanosaur remains in the same formation. It is important to note that none of the aforementioned conditions is exclusive to sauropod dinosaurs, with egg size smaller in all definitive sauropod eggs (Chiappe et al. 1998;Grellet-Tinner et al. 2011), rounded shape shared with hadrosaurs and some theropods (Horner and Makela 1979;Mateus et al. 1997), clutch arrangement different to that of confirmed sauropod eggs (Vila et al. 2010); and coeval distribution of eggs and skeletal remains having been proven wrong in the past (Norell et al. 1994(Norell et al. , 2020. Even so, some similarities between the eggshell of the Argentinean Faveoloolithidae and some Megaloolithidae eggshells still support a sauropod affinity of these eggs including nucleation centers at their bases, rhombohedric acicular calcite crystals, prominent compactituberculate ornamentation (Grellet-Tinner and Fiorelli 2010), although definitive embryos are needed to test this hypothesis. ...
... There, the authors interpreted that babies were still living in the nest at their time of death (Bonaparte and Vince 1979). Four decades after, Norell et al. (2020) concluded, after a detailed examination, that Mussaurus eggs were soft (but see Sect. 13.3.4 ...
Chapter
The South American sauropodomorph egg record is unrivaled in its richness, with Jurassic and Early and Late Cretaceous eggshell occurrences of up to six different oospecies, included in the oofamilies Megaloolithidae, Fusioolithidae and, probably, Faveoloolithidae, and the oldest putative soft-shelled eggs of the fossil record. In addition, numerous clutches, some nests, exquisitely preserved embryos with skin impressions, and delicate embryological structures such as the egg tooth, and perinatal individuals of stem sauropodomorphs have been reported from South America. Thus, it represents one of the most complete oological and developmental records of an extinct clade and provides a unique opportunity to explore its palaeobiology and palaeoecology. In this chapter, the reader will find a detailed revision of the egg-bearing localities of South America, combined with a critical review of all specimens previously referred to as sauropodomorph taxa and parataxa. Detailed account of the impressive sauropodomorph embryo record is provided. Finally, the different Argentinean nesting grounds are discussed in their paleoenvironmental context, to provide an updated picture of the nesting strategies and reproductive traits of South American sauropodomorphs.
... [1,2] Applications of non-destructive, in situ Raman microspectroscopy have resulted in rapid progress in understanding biomolecule fossilization and the detection of biosignatures based on comparative statistical analyses of fossil organic matter. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] The in situ approach facilitates rapid non-destructive analysis of surface-cleaned samples without requiring time-consuming extractions of the organic matter that may alter fossil molecular compounds. Raman spectroscopy not only characterizes molecular functional groups (small molecular units with distinct chemical properties), but also provides insights into higher-level structural organization by detecting intermolecular and organo-mineral interactions. ...
... [19,[24][25][26][27] A number of advantages, including time-efficient non-destructive analysis, availability and low operating cost of equipment, and the utility of results, make Raman and complementary types of light spectroscopy ideal for molecular tests of hypotheses based on the composition of paleontological and geological materials. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][20][21][22][23] A diversity of studies conducted by different laboratories have recovered similar patterns in the molecular makeup of fossil organic matter in independently acquired in situ Raman spectra. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][20][21][22][23] However, a recent Perspective by Alleon et al. [28] concluded that the biological results of a selected subset of these studies [3,4,6,11,12,15] are compromised, based on their detection of sinusoidal edge filter ripples, i.e., instrumental artefacts, in the Raman data. ...
... [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][20][21][22][23] A diversity of studies conducted by different laboratories have recovered similar patterns in the molecular makeup of fossil organic matter in independently acquired in situ Raman spectra. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][20][21][22][23] However, a recent Perspective by Alleon et al. [28] concluded that the biological results of a selected subset of these studies [3,4,6,11,12,15] are compromised, based on their detection of sinusoidal edge filter ripples, i.e., instrumental artefacts, in the Raman data. [28] The results in the disputed studies are based on the statistical evaluation of in situ Raman spectra obtained in the organic fingerprint region (500-1800 cm −1 ) for a diversity of modern, experimentally matured, and fossil tissues covering all major branches of extinct and modern invertebrates and vertebrates, as well as sedimentary host rocks, and represent the first large-scale explorative studies of their kind in the geosciences. ...
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A recent article argued that signals from conventional Raman spectroscopy of organic materials are overwhelmed by edge filter and fluorescence artefacts. The article targeted a subset of Raman spectroscopic investigations of fossil and modern organisms and has implications for the utility of conventional Raman spectroscopy in comparative tissue analytics. The inferences were based on circular reasoning centered around the unconventional analysis of spectra from just two samples, one modern, and one fossil. We validated the disputed signals with in situ Fourier-Transform Infrared (FT-IR) Spectroscopy and through replication with different lasers, filters, and operators in independent laboratories. Our Raman system employs a holographic notch filter which is not affected by edge filter or other artefacts. Multiple lines of evidence confirm that conventional Raman spectra of fossils contain biologically and geologically meaningful information. Statistical analyses of large Raman and FT-IR spectral data sets reveal patterns in fossil composition and yield valuable insights into the history of life.
... The posture appears to be distinct from that of oviraptorids as the skull is located near the pole, and the neck curls dorsally rather than ventrally (Reisz et al., 2010). This is an unusual embryonic head posture, which may be due to the presence of soft-shelled eggs in these dinosaurs (Norell et al., 2020) that lacked the rigidity to maintain the original posture after death. This atypical head posture alternatively could have been due to movements associated with the hatching process. ...
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Birds are the only living amniotes with coloured eggs1-4, which have long been considered to be an avian innovation1,3. A recent study has demonstrated the presence of both red-brown protoporphyrin IX and blue-green biliverdin5-the pigments responsible for all the variation in avian egg colour-in fossilized eggshell of a nonavian dinosaur6. This raises the fundamental question of whether modern birds inherited egg colour from their nonavian dinosaur ancestors, or whether egg colour evolved independently multiple times. Here we present a phylogenetic assessment of egg colour in nonavian dinosaurs. We applied high-resolution Raman microspectroscopy to eggshells that represent all of the major clades of dinosaurs, and found that egg colour pigments were preserved in all eumaniraptorans: egg colour had a single evolutionary origin in nonavian theropod dinosaurs. The absence of colour in ornithischian and sauropod eggs represents a true signal rather than a taphonomic artefact. Pigment surface maps revealed that nonavian eumaniraptoran eggs were spotted and speckled, and colour pattern diversity in these eggs approaches that in extant birds, which indicates that reproductive behaviours in nonavian dinosaurs were far more complex than previously known3. Depth profiles demonstrated identical mechanisms of pigment deposition in nonavian and avian dinosaur eggs. Birds were not the first amniotes to produce coloured eggs: as with many other characteristics7,8 this is an attribute that evolved deep within the dinosaur tree and long before the spectacular radiation of modern birds.
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The cuticle layer consisting mainly of lipids and hydroxyapatite (HAp) atop the mineralized avian eggshell is a protective structure that prevents the egg from dehydration and microbial invasions. Previous ornithological studies have revealed that the cuticle layer is also involved in modulating the reflectance of eggshells in addition to pigments (protoporphyrin and biliverdin). Thus, the cuticle layer represents a crucial trait that delivers ecological signals. While present in most modern birds, direct evidence for cuticle preservation in stem birds and non-avian dinosaurs is yet missing. Here we present the first direct and chemical evidence for the preservation of the cuticle layer on dinosaur eggshells. We analyze several theropod eggshells from various localities, including oviraptorid Macroolithus yaotunensis eggshells from the Late Cretaceous deposits of Henan, Jiangxi, and Guangdong in China and alvarezsaurid Triprismatoolithus eggshell from the Two Medicine Formation of Montana, United States, with the scanning electron microscope (SEM), electron probe micro-analysis (EPMA), and Raman spectroscopy (RS). The elemental analysis with EPMA shows high concentration of phosphorus at the boundary between the eggshell and sediment, representing the hydroxyapatitic cuticle layer (HAp). Depletion of phosphorus in sediment excludes the allochthonous origin of the phosphorus in these eggshells. The chemometric analysis of Raman spectra collected from fossil and extant eggs provides further supportive evidence for the cuticle preservation in oviraptorid and probable alvarezsaurid eggshells. In accordance with our previous discovery of pigments preserved in Cretaceous oviraptorid dinosaur eggshells, we validate the cuticle preservation on dinosaur eggshells through deep time and offer a yet unexplored resource for chemical studies targeting the evolution of dinosaur nesting ecology. Our study also suggests that the cuticle structure can be traced far back to maniraptoran dinosaurs and enhance their reproductive success in a warm and mesic habitat such as Montana and southern China during the Late Cretaceous.
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Fossil eggs and embryos that provide unique information about the reproduction and early growth of vertebrates are exceedingly rare, particularly for pterosaurs. Here we report on hundreds of three-dimensional (3D) eggs of the species Hamipterus tianshanensis from a Lower Cretaceous site in China, 16 of which contain embryonic remains. Computed tomography scanning, osteohistology, and micropreparation reveal that some bones lack extensive ossification in potentially late-term embryos, suggesting that hatchlings might have been flightless and less precocious than previously assumed. The geological context, including at least four levels with embryos and eggs, indicates that this deposit was formed by a rare combination of events, with storms acting on a nesting ground. This discovery supports colonial nesting behavior and potential nesting site fidelity in the Pterosauria.
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The reproductive biology of living birds differs dramatically from that of other extant vertebrates. Although some attributes of modern avian reproduction had their origin within theropod dinosaurs like oviraptors and troodontids, even the most derived non-avian theropods lack key features of modern birds. We review the current knowledge of reproduction in Mesozoic birds and 3 lines of evidence that contribute to our understanding of the evolution of the modern avian reproductive mode: (1) efforts to define the ancestral reproductive condition on the basis of extant birds, (2) the fossil record of non-avian theropod dinosaurs, and (3) the fossil record of reproduction in primitive Mesozoic birds (e.g., Enantiornithes). The fossil evidence from Mesozoic birds and non-avian theropods suggests that reproduction passed through 5 stages from basal theropods to neornithines: (1) pre-maniraptoran theropods, (2) oviraptor-grade maniraptorans, (3) troodontid-grade paravians, (4) Enantiornithes, and (5) basal Neornithes. Major changes occurred incrementally in egg size, shape, and microstructure; in nest form; in incubation method; and in parental care. Reproduction in troodontid theropods concurs with this clade representing the sister taxon to birds. Reproduction in enantiornithine birds included sequential ovulation from a single ovary and oviduct, eggs planted upright within sediments, and incubation by a combination of sediment and attendant adult or eggs fully buried with superprecocial young. Incubation modes of derived non-avian theropods and enantiornithines may have favored paternal care. Significant changes between enantiornithines and neornithines include an additional increase in relative egg size and sediment-free incubation. The latter permitted greater adult-egg contact and likely more efficient incubation. Associated changes also included improved egg shape, egg rotation, and chalazae-the albumin chords that suspend the yolk and facilitate proper embryonic development during rotation. Neornithes are the only Mesozoic clade of Dinosauria to nest completely free of sediment, and this may have played a crucial role in their surviving the K-Pg mass extinction event.
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