Evidence for heterothermic endothermy and reptile-like eggshell mineralization in Troodon, a non-avian maniraptoran theropod.

The dinosaur-bird transition involved several anatomical, biomechanical, and physiological modifications of the theropod bauplan. Non-avian maniraptoran theropods, such as Troodon, are key to better understand changes in thermophysiology and reproduction occurring during this transition. Here, we applied dual clumped isotope (Δ47 and Δ48) thermometry, a technique that resolves mineralization temperature and other nonthermal information recorded in carbonates, to eggshells from Troodon, modern reptiles, and modern birds. Troodon eggshells show variable temperatures, namely 42 and 29 ± 2 °C, supporting the hypothesis of an endothermic thermophysiology with a heterothermic strategy for this extinct taxon. Dual clumped isotope data also reveal physiological differences in the reproductive systems between Troodon, reptiles, and birds. Troodon and modern reptiles mineralize their eggshells indistinguishable from dual clumped isotope equilibrium, while birds precipitate eggshells characterized by a positive disequilibrium offset in Δ48. Analyses of inorganic calcites suggest that the observed disequilibrium pattern in birds is linked to an amorphous calcium carbonate (ACC) precursor, a carbonate phase known to accelerate eggshell formation in birds. Lack of disequilibrium patterns in reptile and Troodon eggshells implies these vertebrates had not acquired the fast, ACC-based eggshell calcification process characteristic of birds. Observation that Troodon retained a slow reptile-like calcification suggests that it possessed two functional ovaries and was limited in the number of eggs it could produce; thus its large clutches would have been laid by several females. Dual clumped isotope analysis of eggshells of extinct vertebrates sheds light on physiological information otherwise inaccessible in the fossil record.

Effects of oxygen plasma ashing treatment on carbonate clumped isotopes.

RATIONALE: For clumped isotope analysis (Δ47 ), hydrocarbon and organic molecules present an important contaminant that cannot always be removed by CO2 purification through a Porapak-Q trap. Low-temperature oxygen plasma ashing (OPA) is a quick and easy approach for treatment; however, the impact of this treatment on the original carbonate clumped isotope values has never been fully studied. METHODS: We tested the isotopic impact of OPA using three natural samples with a large range of initial Δ47 values. Crushed and sieved (125 µm mesh) samples were placed into a Henniker Plasma HPT-100 plasma system and treated at a flow rate of 46 mL/min and a power of 100 W at a vacuum of 0.2 mbar for 10, 20, 30 and 60 min before clumped isotope analysis using two MAT 253 isotope ratio mass spectrometers modified to measure masses 44-49. RESULTS: OPA treatment for 30 min or more on calcite powder samples has the potential to alter the clumped isotopic composition of the samples beyond analytical error. A systematic positive offset is observed in all samples. The magnitude of this alteration translates to a temperature offset from known values ranging from 4°C to 13°C. We postulate that the observed positive offset in Δ47 occurs because the bonds within lighter isotopologues are preferentially broken by plasma treatment, leading to an artificial increase in the 'clumping' value of the sample. CONCLUSIONS: We recommend that any laboratory performing OPA treatments should reduce the runs to 10-20 min or carry out successive runs of 10 min followed by sample stirring, as this procedure showed no alteration in the initial Δ47 values. Our results validate the use of OPA for clumped isotope applications and will allow future research to use clumped isotopes for challenging samples such as oil-stained carbonates, bituminous shales or host rocks with very high organic carbon content.

Reducing contamination parameters for clumped isotope analysis: The effect of lowering Porapak™ Q trap temperature to below -50°C.

RATIONALE: Carbonate clumped isotope thermometry examines the thermodynamic preference of 13 C-18 O bonds to form within the carbonate crystal lattice. The 13 C18 O16 O isotopologue in analyte CO2 has a natural abundance of 44.4 ppm necessitating stringent purification procedures to remove contaminant molecules that may produce significant isobaric effects within range of the mass 47 isotopologue. Strict purifications of analyte CO2 are thus required as well as reliable contamination indicators. METHODS: CO2 purification was carried out by vacuum cryogenic purification through a static trap packed with Porapak™ Q (PPQ). The correlation between mass excesses on m/z 47, 48 and 49 in CO2 produced by acid digestion of 12 natural samples was measured by isotope ratio mass spectrometry (IRMS). CO2 from two contaminated carbonate samples was then purified at PPQ trap temperatures between -25 and -65°C and measured by IRMS to determine changes in mass excesses on m/z 47, 48 and 49. Finally carbonate standards, Carrara marble (CM) and ETH3, were purified at PPQ trap temperatures of -35 and -60°C to identify isotopic fractionation associated with lowering trap temperature. RESULTS: The correlation between mass excesses on m/z 47, 48 and 49 is determined to be sample dependent. Lowering the PPQ trap temperature to -60°C has a 78% success rate in decreasing Δ48offset , a measure of sample contamination, to within an acceptable range (<1.5 ‰). Lowering the PPQ temperature in purification of CM and ETH3 is associated with decreases in the δ13 C and δ18 O values as a result of isotopic fractionation. We demonstrate that we can correct for fractionation at a trap temperature of -60°C. CONCLUSIONS: Lowering the temperature of the Porapak Q trap to -60°C results in improved sample cleaning. It is possible to correct for fractionation in δ13 C and δ18 O values at lower PPQ trap temperatures using identically prepared standards. This result has important connotations for laboratories using similar sample preparation methods.