Materials and Methods Extant eggshell material The emu eggshells (Dromaius novaehollandiae) were produced by captive birds and are stored in the ZFMK collections (ZFMK uncat.). Emu eggshell is reported to contain minimal amounts of PP in addition to some of the highest naturally occurring amounts of BV (Gorchein, Lim & Cassey, 2009). Fossil eggshell material We sampled three oviraptorid Macroolithus yaotunensis eggs from the collections of the NMNS and STIPB covering three geographically and taphonomically distinct Chinese deposits. Investigated specimens were collected in the Liguanqiao Basin near Nanyang in the province of Henan (STIPB E54/1), from the Hongcheng Basin in the province of Jiangxi (NMNS CYN-2004-DINO-05/I), and the Nanxiong Basin in province of Guangdong (STIPB E54/3). Detailed descriptions of the localities in context of geological and taphonomic settings are included in the Supplemental Information. Macroscopically, all oviraptorid eggshell samples had a blackish to blackish-brownish (after cleaning them from adherent sediment) color, revealing a very subtle shimmer of blue–green at angled light conditions. Historically, samples derived from the Liguanqiao Basin are Late Cretaceous in age and derived from the fluvial/alluvial deposits (red sandstones) of the Hugang Formation. They have been housed in STIPB since 1983 and were previously described by Erben (1995). Preserved oviraptorid eggs from the Hongcheng Basin in the province of Jiangxi were obtained from the Late Cretaceous Tangbian Formation which comprises fluvial red sandstones. The Hongcheng Basin and the Nanxiong Basin may belong to the same extended basin complex (Liu, 1999). The Late Cretaceous strata of the Nanxiong Basin are divided into the Yuanpu Formation and the overlying Pingling Formation. The Yuanpu Formation, which might be correlated with the fossil-rich Mongolian Djadochta Formation, yielded our eggshell samples from alluvial sediments (red silt-sandstones), and is dated as Maastrichtian in age (Zhao et al., 1991). One of two preserved complete eggs (Fig. 1A) from the Chinese province of Jiangxi (NMNS CYN-2004-DINO-05) which were previously assigned to the oviraptorid egg parataxon Macroolithus yaotunensis was sampled over four zones of the egg (Figs. 1B and 2B), prepared for histology, and then used for porosity measurements (see Supplemental Information). These four zones represent the blunt, middle, and acute parts of the egg, and were separated to approach zonal differences in porosity values which were tested for maximum porosity at the mid portions to indicate egg storage in an open nest (based on Varricchio et al., 2008). Measured porosity values were compared to published dinosaur and avian porosity patterns and used to calculate the eggshell water vapor conductivity. Samples for chemical analyses were taken separately. Sediment adhering to the complete eggs (NMNS CYN-2004-DINO-05/I) was sampled additionally to confirm that we are not dealing with wholesale sample contamination. A single sediment sample (red silty sandstone) was available to test against wholesale contamination with BV and PP of the sample since only the complete oviraptorid eggs from the province of Jiangxi provided original, attached matrix sediment. The two-remaining fossil oviraptor eggshell samples represent isolated fragments freed of original matrix. Figure 1: Provenance of Heyuannia eggshell, reconstructed zonal egg water vapor conductance, oviraptor clutch structure, and corrected, reconstructed egg color. Heyuannia huangi egg (NMNS CYN-2004-DINO-05/I) calculated from BV and PP concentrations and porosity measurements (see (A) Geographical map of China. The capital city, Beijing, is indicated by the red star. Red shaded provinces indicate the three different localities where the specimens were collected: the Liguanqiao Basin in Henan, the Hongcheng Basin in Jiangxi, and the Nanxiong Basin in Guangdong (see Supplemental Information ). (B) The reconstructed color and average zonal water vapor conductance of the left Jiangxiegg (NMNS CYN-2004-DINO-05/I) calculated from BV and PP concentrations and porosity measurements (see Supplemental Information ). (C) Top view of an oviraptor clutch (PFMM 0010403018). This clutch illustrates how eggs are arranged in pairs with their blunt ends pointing to the clutch center. The eggs are arranged in layers separated by sediment. (D) Reconstruction of a partially open oviraptorid nest. Note that the original inclination of the eggs would have been steeper than their preserved attitude (C) due to sediment compaction. Figure 2: (A) Pair of oviraptorid Heyuannia eggs (NMNS CYN-2004-DINO-05) from the Chinese province of Jiangxi before sampling. Porosity measurements and calculations of water vapor conductance are based on these eggs. Pieces of eggshell from each of the four zones depicted in (B) were used in porosity measurements. (B) Egg model separated into four zones used for zonal porosity measurements. Therefore, double half-prolate spheroids and cone models of the idealized egg were used to estimate the zonal surface areas to eventually approximate water vapor conductance. Zone 1 represents the blunt end of the egg, zones 2 and 3 the mid portions, and zone 4 represents the pointed end of the egg.

Methods We used two commercial standards (biliverdin dihydrochloride and protoporphyrin IX, purchased from Sigma Aldrich), one extant bird eggshell sample (emu), the three fossil Heyuannia huangi eggshell samples, and one sediment sample (reddish sandstone) for High Performance Liquid Chromatography coupled to Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry (HPLC ESI Q-ToF MS). The basis for this sample selection was (1) to demonstrate reproducibility in three fossil eggshell samples, (2) to exclude the possibility of wholesale contamination due to sample or system exogenous PP or BV input by analyzing sediment adhering to a sample of eggshell, (3) to provide a sensitivity control and quality marker of the analytical routine based on precise detection and quantification of emu eggshell (ranging from the upper (BV) to the lower (PP) detection limit in terms of pigment concentrations), and (4) to generate a calibrated concentration signal for quantification based on the known concentrations of the commercial standards. Adhering sediment and other superficial contaminations were chemically removed from all eggshell samples by a boosted decalcification of the outermost sample surfaces. 500 µL of disodium EDTA solution (100 mg/mL), adjusted to a pH of 7.2, were added to the 180–562 mg eggshell samples and sediment control sample, each of which was stored in 1 mL Eppendorf tube. Samples were incubated for 5 min and then transferred to fresh Eppendorf tubes. The decalcification residue was discarded. Pigment decalcification was performed immediately after preliminary cleaning of the samples. The three oviraptorid eggshell samples, the emu eggshell, and the sediment control sample were incubated again in the EDTA solution which was already used for initial cleaning, this time for 5 min. During this 5 min, the sample tubes were vortexed three times for 1 min. Vortexing was performed with uncapped tubes to allow outgassing of the carbon dioxide generated. After 5 min of incubation in the EDTA decalcification solution, all sample tubes were centrifuged at 15,000 g for 1 min. After centrifuging, the supernatant solutions were collected in separate tubes, while the decalcified sample precipitates were filled up with fresh EDTA solution from the stock. Incubation of 5 min followed, including vortexing 3× in uncapped tubes, as in the previous step. The samples were centrifuged again at 15,000 g for 1 min, supernatants were collected, and the precipitates filled up with fresh EDTA solution. As in the previous step, incubation of 5 min including vortexing of the uncapped sample tubes followed. After a final round of centrifuging for 1 min, supernatant solutions were collected, and the partially decalcified precipitates were used for the final pigment extraction. 1 mL of acetonitrile/acetic acid (4:1, v/v) was added to the decalcified sample pellets for 10 min of incubation, including 2 min of vortex-mixing. Afterwards the sample tubes were centrifuged at 15,000 g for 2 min, and the supernatant solution holding the pigment extract were transferred into fresh Eppendorf tubes, and stored in a dark environment at 4 °C. The commercial standards were dissolved in the same acetonitrile-acetic acid solution (4:1, v/v) and stored with the sample pigment extracts. The filtered extracts and commercial standard solutions were stored less than 24 h before they were injected into an HPLC Dionex Ultimate 3000 (Thermo Scientific) separating sample compounds by using a EC50/2 Nucleodur C18 Gravity 3 µm column (Macherey-Nagel). Reverse-phase HPLC was run at a flow rate of 0.3 mL/min. HPLC was started at 90% H 2 O containing 0.1% acetic acid. The gradient started after 1 min and reached 100% acetonitrile after 14 min. For an additional 7 min, the column was flushed with 100% acetonitrile (containing 0.1% acetic acid). For the biliverdin analysis, 15 µL sample solution was injected, and 20 µL for the protoporphyrin detection. 2 min of washing runs between each sample extract cleaned the entire system. The liquid chromatography system was coupled to a micrOTOF-Q mass spectrometer (Bruker) with an electrospray ionization (ESI) source inducing positive ionization. Data were collected in positive full scan MS mode over the range of 50–1,000 m/z, using a capillary voltage of 4.5 kV and an end plate offset of −500 V. The dry heater of the ESI source was set at 200 °C. Nitrogen desolution and nebulizer gas flow was 10.0 L/min; the nebulizer was run at 2.2 bar. Time-of-Flight (TOF) detection allowed the determination of the accurate masses of biliverdin and protoporphyrin. Figure 3: ESI (+) MS extracted ion chromatograms (EICs) for mass 583.2520 ± 0.01/0.05 m/z, indicative of BV, and mass 563.2653 ± 0.01/0.05 m/z, indicative of PP. We identified BV and PP by retention time, exact mass and isoform/tautomer separation. EICs for 583.2520 ± 0.01/0.05 m/z are depicted for the commercial BV standard, emu eggshell, extracts of Heyuannia huangi eggshells derived from the Chinese provinces Henan, Jiangxi, and Guangdong and the sediment control extract from Jiangxi. Unmodified BV elutes after 8 min retention time, and was proven present for the biliverdin commercial standard, the emu eggshell, and the three oviraptorid eggshells. The sediment sample was used as control for contamination, and its EIC does not show a peak after 8 min retention time, proving the absence of biliverdin in the sediment sample and the originality of biliverdin detected for the eggshell samples. EICs for 563.2653 ± 0.01/0.05 m/z are depicted for the commercial PP standard, emu eggshell, Heyuannia huangi eggshell derived from the Chinese provinces Henan, Jiangxi, and Guangdong, and the sediment sample from Jiangxi. Unmodified PP elutes after 14 min and was proven present for the PP commercial standard, the emu eggshell, and the fossil oviraptorid eggshells. There is no peak in the PP EIC for the sediment sample after 14 min, what proves the absence of PP in the sediment control sample, and the originality of PP in the eggshell samples. Peak intensity correlates with pigment concentrations in the extracts.

Results We reliably identified both BV, as [M + H]+ with 583.2520 m/z (calculated mass: 583.2551 g/mol) after 8 min retention time, and PP, as [M + H]+ complex with 563.2623 m/z (calculated mass: 563.2653 g/mol) after 14 min retention time (Fig. 3A) in the commercial standard solutions, the emu eggshell, and the three fossil oviraptorid eggshell samples. The commercial standard solutions of known concentrations were used to identify the chemotrait-specific, diagnostic retention times for BV and PP on the chromatographic column. Elution of BV was consistent after 8 min, while elution of PP delayed consistently until 14 min of the run mobile phase gradient (consistent with Igic et al., 2009). A second compound-diagnostic trait was provided by ionization after elution from the chromatographic column, followed by exact mass determination of the [M + H]+ ion complexes. Differing behavior of BV and PP native to eggshells compared to commercially purified BV and PP was ruled out by identical retention times, number of isoforms/tautomers, and exact mass peaks of emu eggshell BV and PP and the commercial standard solutions. Furthermore, maximum sensitivity of the HPLC ESI MS system was demonstrated by precise detection of pigment concentrations in the emu eggshell solution which approached the upper (BV) and the lower (PP) detection limits. The extracted ion chromatograms (EICs) for BV and PP obtained from the sediment sample whole ion mass spectrum yielded signals within broader tolerances of the BV and PP exact masses, but no peaks corresponding to the commercial standard calibrated retention times were identified. Absence of a diagnostic mass peak after 8 min retention time on the column for BV, and after 14 min retention time on the column for PP, is a significant demonstration of absence of trace amounts BV and PP in the sediment sample. Thereby, contamination of the samples or the detection system was excluded and originality of the detected pigments in the oviraptorid eggshell samples is guaranteed. Quantification of the detected pigment concentrations based on commercial standard calibration was determined by application of an experiment-empirical correction for the extraction loss of BV due to its increased hydrophily of the fossil oviraptor eggs. We found the highest preserved concentrations of BV in the eggshells from Henan (6 nmol/g), followed by the eggshells from Jiangxi (2 nmol/g) and those from Guangdong (1 nmol/g). The preserved PP concentrations ranged from 2 nmol/g in both the Henan and Jiangxi eggshells to 1 nmol/g in the Guangdong eggshells. Our empirical correction applied to the fossil eggshell samples yielded very realistic pigment concentration estimates for the emu eggshell of 2 nmol/g PP, and 266 nmol/g BV which fall into the reported range of emu eggshell pigment concentrations in the scientific literature (Table S1). The bluish shimmer of the fossil oviraptorid eggshells suggests generally higher BV concentrations than those we detected, as also found in a similar study on pigment preservation in subfossil moa eggshells using the same methodology. The color of the fossil eggs suggested higher BV concentrations than the authors managed to detect. Taken together, our study and the study by Igic et al. (2009) imply that bluish or greenish coloring degradation products of BV remain which therefore shows slightly different chemical properties and different exact masses, and is thus not detected by an LC MS system targeting unmodified compounds measurable against commercial standards. To demonstrate the perceivability of a visual color signal based on the detected pigment concentrations in the oviraptorid eggshells, we plotted our pigment concentrations into the comprehensive pigment concentration-color matrix of Cassey et al. (2012). Our three dinosaur egg color data points fall in the visibly olive-green color range between Haliaetus albicilla and Circus aeruginosus (Cassey et al., 2012). They plot in the cluster of unspotted eggs, suggesting an immaculate, homogenous coloration. No patterns were visible in the fossil eggs (Fig. 1). Since the preserved fossil oviraptor eggshell color suggests originally higher BV concentrations, taphonomy needs to be considered to generate a realistic, native oviraptor egg color reconstruction. Because BV is more reactive and more hydrophilic, and thus soluble in sediment-percolating aqueous fluids, the concentrations of unmodified, preserved pigments after at least 66 million years of sedimentary burial are much more likely to be significantly lowered than those of the more stable, hydrophobic PP (Falk, 1964). Therefore, the taphonomic projections of our preserved pigment concentrations in the avian egg color space (Cassey et al., 2012) realistically lift the investigated oviraptor egg colors significantly towards much higher BV values, while the shift towards increased PP values would be only minimal. However, our fossil oviraptor eggs would remain deeply nested within the area of unspotted eggs (based on Cassey et al., 2012). Such an additional taphonomic correction of the reconstructed egg color approximates an intensively blue–greenish oviraptorid egg color. Whether the differences in preserved pigment concentrations between the three fossil oviraptor egg samples from different localities reflect intraspecific variation in egg color or different taphonomic conditions in the deposits cannot be reliably assessed at this point and requires future investigations. Since these differences in preserved pigment concentrations in the oviraptorid eggshells affect the BV values much stronger (range 6–1 nmol/g) than they affect the PP values (range 2–1 nmol/g), we assume that differences in color are more likely to be taphonomic. In vivo intraspecific variation of egg color would most likely affect BV and PP concentrations equally, while taphonomic effects affect BV concentrations much stronger than PP concentrations (Falk, 1964). Also, the original egg color is overprinted by a generally blackish-brownish hue (Fig. 1). This brownish discoloration traces back to preserved, oxidatively crosslinked eggshell organic matrix proteins of the AGE/ALE-type (Wiemann et al., 2016). Our reconstruction of colored eggs for oviraptors is consistent with our reevaluation of the oviraptorid nesting mode: we consider oviraptor eggs as lying at least partially open in the nest (consistent with (Norell et al., 1995)). This reconstruction is based on the estimated water vapor conductance of 108.66 mg H 2 O day−1 Torr−1 for the Heyuannia egg NMNS CYN-2004-DINO-05/I from the province of Jiangxi (Fig. 1A). This value is calculated from the four zonal conductances deduced from zonal porosity counts (Supplemental Information). The highest values for shell porosity were found in the middle portion of the oviraptor egg (zones 2 and 3), and especially pronounced in zone 3 with a resultant conductance of 43.6 mg H 2 O day−1 Torr−1 (Table S5). The pointed end (zone 4) which is stuck in the nest material, has a calculated conductance of 22.88 mg H 2 O day−1 Torr−1, while the exposed blunt end (zone 1) has a calculated conductance of 13.77 mg H 2 O day−1 Torr−1 (Table S5).

Conclusions Our study extends the origin of colored eggs from crown birds to oviraptorid dinosaurs. The result has important implications both for the origin of avian biology and the reproductive biology of theropods dinosaurs. This work also broadens the scope of paleontological research on molecular preservation and ecology to hard vertebrate tissues. Our study ties together previous hypotheses on the eumaniraptoran origin of partially open nesting, and paternal care. Also, potential future avenues for investigation are posed by the potential linkage between blue–green egg color and communal nesting, as well as polyandry, which represent yet unaddressed topics in extinct archosaurs. The second aspect of our work focuses on its implications for molecular and soft tissue preservation through deep time. Chemically stable, relatively small biological molecules such as PP and BV appear to be protected from complete degradation over millions of years in carbonate biomineral matrices, in an oxidative sediment milieu. Similar biomolecule preservation may also be present in enamel, dentine and bone mineral. Ancient biomolecules and the soft tissues which they construct pave the way to trace life and its behaviors through time and, thus, invite further studies since they are easily detectable, more abundant than expected, and revolutionary in their ecological implications.