1. Biochemistry and Chemical Biology
  2. Microbiology and Infectious Disease
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Cretaceous dinosaur bone contains recent organic material and provides an environment conducive to microbial communities

  1. Evan T Saitta  Is a corresponding author
  2. Renxing Liang
  3. Maggie CY Lau
  4. Caleb M Brown
  5. Nicholas R Longrich
  6. Thomas G Kaye
  7. Ben J Novak
  8. Steven L Salzberg
  9. Mark A Norell
  10. Geoffrey D Abbott
  11. Marc R Dickinson
  12. Jakob Vinther
  13. Ian D Bull
  14. Richard A Brooker
  15. Peter Martin
  16. Paul Donohoe
  17. Timothy DJ Knowles
  18. Kirsty EH Penkman
  19. Tullis Onstott
  1. Field Museum of Natural History, United States
  2. Princeton University, United States
  3. Chinese Academy of Sciences, China
  4. Royal Tyrrell Museum of Palaeontology, Canada
  5. University of Bath, United Kingdom
  6. Foundation for Scientific Advancement, United States
  7. Revive and Restore, United States
  8. Johns Hopkins University, United States
  9. American Museum of Natural History, United States
  10. Newcastle University, United Kingdom
  11. University of York, United Kingdom
  12. University of Bristol, United Kingdom
Research Article
Cite this article as: eLife 2019;8:e46205 doi: 10.7554/eLife.46205
69 figures, 30 tables, 1 data set and 1 additional file

Figures

Light microscopy (A–C) and VPSEM (D–G) images and EDS spectra (H–M) of HCl demineralized, freeze-dried samples.

(A–C) samples rested on carbon tape upon SEM stubs and the pitting was a result of prior VPSEM and EDS analysis. (A) Centrosaurus vessels and associated minerals. (B, F, L) Carcharias tooth. (C, G, M) Gallus. (D) infilled Centrosaurus vessel. (E) Centrosaurus vessel, fibrous material along the center of the vessel, and associated reddish minerals around the vessel. (H) Centrosaurus vessel exterior from D. (I) Centrosaurus vessel infilling from D. (J) associated reddish mineral in Centrosaurus. (K) Centrosaurus fibrous material from E. Centrosaurus samples are matrix-surrounded subterranean bone.

https://doi.org/10.7554/eLife.46205.003
ATR FTIR spectra of HCl demineralized, freeze-dried samples.

(A) Gallus. (B) Carcharias tooth. (C) matrix-surrounded subterranean Centrosaurus bone vessel with inset showing a composite image of the vessel that was analyzed.

https://doi.org/10.7554/eLife.46205.004
Py-GC-MS total ion chromatograms of samples ethanol rinsed before powdering.

Some of the major pyrolysis products are labeled with the compound name or prominent m/z peaks. (A) Gallus bone. (B) Matrix-surrounded subterranean Centrosaurus bone. (C) Adjacent mudstone matrix of subterranean Centrosaurus bone in B. (D) Humic acid (technical grade) powder with a series of branched and cyclic alkanes, several aromatic ions, and several hopanoid (m/z = 191, 189, 367) and steroid (m/z = 217, 129, 257) ions.

https://doi.org/10.7554/eLife.46205.005
Py-GC-MS chromatograms searching for ion m/z ranges typical of n-alkanes and n-alkenes from kerogen in the matrix-surrounded subterranean Centrosaurus bone ethanol rinsed before powdering.

Potential doublets indicative of n-alkanes/n-alkenes are weakly apparent at best. A, m/z = 55. B, m/z = 57. C, m/z = 83. D, m/z = 85.

https://doi.org/10.7554/eLife.46205.006
Py-GC-MS chromatograms searching for ion m/z ranges typical of n-alkanes and n-alkenes from kerogen in the uncovered subterranean Centrosaurus bone ethanol rinsed before powdering.

Doublets indicative of n-alkanes/n-alkenes are relatively more abundant than in Figure 4. A, m/z = 55. B, m/z = 57. C, m/z = 83. D, m/z = 85..

https://doi.org/10.7554/eLife.46205.007
THAA compositional profiles of the KOH-treated samples based on amino acid percentages.

Lines connecting points are added to aid visualization. (A) Late Cretaceous subterranean bone compared to non-aseptically collected Pleistocene-Holocene teeth (with a repeated measurement for the ethanol rinsed sample) and modern bone. (B) Late Cretaceous subterranean bone compared to surface-eroded Late Cretaceous bone from the same outcrop. (C) Surface-eroded Late Cretaceous bone compared to Pleistocene-Holocene teeth and modern bone. (D) Late Cretaceous subterranean bone aseptically collected compared to the adjacent mudstone matrix. (E) Surface-eroded Late Cretaceous bone compared to topsoil at higher elevation (i.e., prairie level) on the same ridge. (F) PCA on non-normalized amino acid percentages (i.e. percentages that do not require further normalization) (see A–E legends). PC1 and PC2 describe 55.04% and 22.66% of the data variation, respectively. See Appendix 1 (Appendix 1—figure 21; Appendix 1—table 9) for PCA summary. Color and symbol coding is constant throughout.

https://doi.org/10.7554/eLife.46205.008
THAA concentrations (summed total of all amino acids measured) of the KOH-treated samples.

(A) Logarithmic scale comparison of modern bone, matrix-surrounded subterranean Late Cretaceous bone, Pleistocene-Holocene surface-eroded teeth (with a repeated measurement for the ethanol rinsed sample), and topsoil on same ridge and ~64 m above BB180. (B) Comparison between fossil Late Cretaceous bone and mudstone.

https://doi.org/10.7554/eLife.46205.009
Microscopic images of EDTA demineralized, PI stained matrix-surrounded subterranean Centrosaurus bone.

(A–B) Fibrous material. (C–D) Vessel. (A, C) Transmission light. (B, D) Fluorescence.

https://doi.org/10.7554/eLife.46205.012
Comparison of microbial community (class level) from matrix-surrounded subterranean Centrosaurus bone and adjacent mudstone matrix.

There are two replicates per sample. Classes with <1% representation in all replicates and samples are combined into an ‘other’ category.

https://doi.org/10.7554/eLife.46205.014
Appendix 1—figure 1
Decay curve of average human proteome based on a half life of 600 years as described by the equation in the text.
https://doi.org/10.7554/eLife.46205.018
Appendix 1—figure 6
Amino acid epimerisation data in Armstrong et al. (1983).
https://doi.org/10.7554/eLife.46205.023
Appendix 1—figure 7
Amino acid concentration data in Armstrong et al. (1983).
https://doi.org/10.7554/eLife.46205.024
Appendix 1—figure 8
Ridge on which BB180 is located. View looking east at mouth of Jackson Coulee, Dinosaur Provincial Park, AB
https://doi.org/10.7554/eLife.46205.026
Appendix 1—figure 9
Region of BB180 sampled prior to removal of overburden.
https://doi.org/10.7554/eLife.46205.027
Appendix 1—figure 10
Region of BB180 sampled after removal of overburden.
https://doi.org/10.7554/eLife.46205.028
Appendix 1—figure 11
Exposed end of Centrosaurus rib upon initial discovery.
https://doi.org/10.7554/eLife.46205.029
Appendix 1—figure 12
Exposed end of Centrosaurus tibia upon initial discovery.
https://doi.org/10.7554/eLife.46205.030
Appendix 1—figure 13
Foil placed on top of sediment and matrix-surrounded Centrosaurus tibia portion prior to flipping with an awl.

Uncovered distal end of tibia is visible to the right of foil.

https://doi.org/10.7554/eLife.46205.031
Appendix 1—figure 14
Centrosaurus tibia after matrix-surrounded sample and uncovered distal end were collected.
https://doi.org/10.7554/eLife.46205.032
Appendix 1—figure 15
Surface eroded bone fragments from BB180 as they were found.
https://doi.org/10.7554/eLife.46205.033
Appendix 1—figure 16
Mudstone from overburden-removed area of BB180 after sampling.
https://doi.org/10.7554/eLife.46205.034
Appendix 1—figure 17
Mudstone collected from 709 m elevation after sampling.
https://doi.org/10.7554/eLife.46205.035
Appendix 1—figure 18
Mudstone from 693 m elevation after sampling.
https://doi.org/10.7554/eLife.46205.036
Appendix 1—figure 19
Surface eroded bone fragments from 691 m elevation as they were found.
https://doi.org/10.7554/eLife.46205.037
Appendix 1—figure 20
Aseptic collection of fossil samples in BB180 on July 8, 2016.

Only skin of the face, wrists, and shins was exposed above the fossils. Shins remained uncovered to act as a thermal window for health and safety reasons to avoid overheating and to reduce the likelihood of contamination by lowering body temperature to reduce perspiration that might fall onto the samples. The body was positioned downhill of the bones at all times to compensate. Photograph by Kentaro Chiba.

https://doi.org/10.7554/eLife.46205.038
Appendix 1—figure 21
Non-normalised PCA biplot of THAA composition of only the sufficiently treated samples associated with the plot in Figure 6F.
https://doi.org/10.7554/eLife.46205.045
Appendix 1—figure 22
Normalised PCA of THAA composition of only the sufficiently treated samples.

prcomp() function in R scale set to ‘TRUE’. Color and shape coding identical to that in Appendix 1—figure 24.

https://doi.org/10.7554/eLife.46205.047
Appendix 1—figure 23
Normalised PCA biplot of THAA composition of only the sufficiently treated samples associated with Appendix 1—figure 22.
https://doi.org/10.7554/eLife.46205.048
Appendix 1—figure 24
THAA compositional profiles of the samples based on amino acid percentages.

(A) Late Cretaceous subterranean bone (red) compared to non-aseptically collected Pleistocene-Holocene teeth (black) and modern bone (blue). (B) Late Cretaceous subterranean bone (red) compared to surface-eroded Late Cretaceous bone from the same outcrop (purple). (C) surface-eroded Late Cretaceous bone (purple) compared to Pleistocene-Holocene teeth (black) and modern bone (blue). (D) Late Cretaceous subterranean bone aseptically collected (red) compared to the adjacent mudstone matrix (brown). (E) surface-eroded Late Cretaceous bone (purple) compared to topsoil at higher elevation (i.e., prairie level) on the same ridge (green). (F) PCA on normalised amino acid percentages (see A–E legends). See Appendix 1 (Appendix 1—figure 25; Appendix 1—table 9) for PCA summary. Color and symbol coding is constant throughout.

https://doi.org/10.7554/eLife.46205.050
Appendix 1—figure 25
Normalised PCA biplot of THAA composition associated with the plot in Appendix 1—figure 24F.
https://doi.org/10.7554/eLife.46205.051
Appendix 1—figure 26
Non-normalised PCA of THAA composition.

prcomp() function in R scale set to ‘FALSE’. Color and shape coding identical to that in Appendix 1—figure 24.

https://doi.org/10.7554/eLife.46205.053
Appendix 1—figure 27
Non-normalised PCA biplot of THAA composition associated with Appendix 1—figure 26.

prcomp() function in R scale set to ‘FALSE’.

https://doi.org/10.7554/eLife.46205.054
Appendix 1—figure 28
THAA concentrations (summed total of all amino acids measured) of the samples.

(A) logarithmic scale comparison of modern Gallus bone (blue), matrix-surrounded subterranean Centrosaurus bone (red), Pleistocene-Holocene surface-eroded shark teeth (black, with a repeated measurement for the ethanol rinsed sample), and topsoil on same ridge and ~64 m above BB180 (green). (B) comparison between fossil Late Cretaceous bone and mudstone. Matrix-surrounded subterranean Centrosaurus bone (solid red), adjacent mudstone matrix of subterranean Centrosaurus bone (solid brown), uncovered subterranean Centrosaurus bone (open red), BB180 mudstone (open brown), surface-eroded Centrosaurus bone from BB180 (solid purple), mudstone on same ridge and ~39 m above BB180 (open tan), mudstone on same ridge and ~23 m above BB180 (solid tan), and surface-eroded Late Cretaceous bone on same ridge and ~21 m above BB180 (open purple). Gelated replicates likely provide the most accurate measurements given the peak reduction present in the non-gelated replicates.

https://doi.org/10.7554/eLife.46205.056
Appendix 1—figure 29
Demineralised modern chicken bone viewed at a distance.
https://doi.org/10.7554/eLife.46205.058
Appendix 1—figure 30
Demineralised modern chicken bone viewed close up (image 1).
https://doi.org/10.7554/eLife.46205.059
Appendix 1—figure 31
Demineralised modern chicken bone viewed close up (image 2).
https://doi.org/10.7554/eLife.46205.060
Appendix 1—figure 32
Demineralised Pleistocene-Holocene shark tooth (image 1).
https://doi.org/10.7554/eLife.46205.061
Appendix 1—figure 33
Demineralised Pleistocene-Holocene shark tooth (image 2).
https://doi.org/10.7554/eLife.46205.062
Appendix 1—figure 34
Mineral grain from demineralised Late Cretaceous Centrosaurus bone.
https://doi.org/10.7554/eLife.46205.063
Appendix 1—figure 35
Vessel from demineralised Late Cretaceous Centrosaurus bone (image 1).
https://doi.org/10.7554/eLife.46205.064
Appendix 1—figure 36
Vessel from demineralised Late Cretaceous Centrosaurus bone (image 2).
https://doi.org/10.7554/eLife.46205.065
Appendix 1—figure 37
Vessel from demineralised Late Cretaceous Centrosaurus bone (image 3).
https://doi.org/10.7554/eLife.46205.066
Appendix 1—figure 38
Electron image of modern demineralised chicken bone full area 1 EDS analysis.
https://doi.org/10.7554/eLife.46205.067
Appendix 1—figure 39
EDS spectrum of demineralised modern chicken bone full area 1.
https://doi.org/10.7554/eLife.46205.068
Appendix 1—figure 40
Electron image of demineralised Pleistocene-Holocene shark tooth full area 1 EDS analysis.
https://doi.org/10.7554/eLife.46205.070
Appendix 1—figure 41
EDS spectrum of demineralised Pleistocene-Holocene shark tooth full area 1.
https://doi.org/10.7554/eLife.46205.071
Appendix 1—figure 42
Electron image of mineral grain from demineralised Late Cretaceous Centrosaurus bone area A.
https://doi.org/10.7554/eLife.46205.073
Appendix 1—figure 43
EDS spectrum of mineral grain from demineralised Late Cretaceous Centrosaurus bone area A.
https://doi.org/10.7554/eLife.46205.074
Appendix 1—figure 44
Electron image of vessel (spot 1) and fibrous mass (spot 2) from demineralised Late Cretaceous Centrosaurus bone area B.
https://doi.org/10.7554/eLife.46205.076
Appendix 1—figure 45
EDS spectrum of vessel (spot 1) from demineralised Late Cretaceous Centrosaurus bone area B.
https://doi.org/10.7554/eLife.46205.077
Appendix 1—figure 46
EDS spectrum of fibrous mass (spot 2) from demineralised Late Cretaceous.

Centrosaurus bone area B.

https://doi.org/10.7554/eLife.46205.079
Appendix 1—figure 47
Electron image of another vessel from demineralised Late Cretaceous Centrosaurus bone area C.
https://doi.org/10.7554/eLife.46205.081
Appendix 1—figure 48
EDS spectrum of vessel from demineralised Late Cretaceous Centrosaurus bone area C.
https://doi.org/10.7554/eLife.46205.082
Appendix 1—figure 49
Electron image of a different vessel exterior (region 1) and interior (region 2) from demineralised Late Cretaceous Centrosaurus bone area D.
https://doi.org/10.7554/eLife.46205.084
Appendix 1—figure 50
EDS spectrum of vessel exterior (region 1) from demineralised Late Cretaceous Centrosaurus bone area D.
https://doi.org/10.7554/eLife.46205.085
Appendix 1—figure 51
EDS spectrum of vessel interior (region 2) from demineralised Late Cretaceous Centrosaurus bone area D.
https://doi.org/10.7554/eLife.46205.087
Appendix 1—figure 52
Electron image of mineral grain from demineralised Late Cretaceous Centrosaurus bone area E with spot one analysis shown.
https://doi.org/10.7554/eLife.46205.089
Appendix 1—figure 53
EDS spectrum of mineral grain from demineralised Late Cretaceous Centrosaurus bone area E, spot 1.
https://doi.org/10.7554/eLife.46205.090
Appendix 1—figure 54
EDTA demineralised Late Cretaceous surface eroded fossil bone at 691 m elevation in Dinosaur Provincial Park showing possible cell clusters as might be expected in a biofilm (image 1).

Sample ID as in Appendix 1—table 2: 16B.

https://doi.org/10.7554/eLife.46205.096
Appendix 1—figure 55
EDTA demineralised Late Cretaceous surface eroded fossil bone at 691 m elevation in Dinosaur Provincial Park showing possible cell clusters as might be expected in a biofilm (image 2).

Sample ID as in Appendix 1—table 2: 16B.

https://doi.org/10.7554/eLife.46205.097
Appendix 1—figure 56
EDTA demineralised Late Cretaceous surface eroded fossil bone at 691 m elevation in Dinosaur Provincial Park showing possible cell clusters as might be expected in a biofilm (image 3).

Sample ID as in Appendix 1—table 2: 16B.

https://doi.org/10.7554/eLife.46205.098
Appendix 1—figure 57
EDTA demineralised Late Cretaceous surface eroded fossil bone at 691 m elevation in Dinosaur Provincial Park showing possible cell clusters as might be expected in a biofilm (image 4).

Sample ID as in Appendix 1—table 2: 16B.

https://doi.org/10.7554/eLife.46205.099
Appendix 1—figure 58
16S rRNA amplicon sequence diversity at the species level of matrix-surrounded subterranean Late Cretaceous Centrosaurus bone and adjacent mudstone matrix.

There are two replicates per sample. Sample ID’s are as in Appendix 1—table 2: B = matrix surrounded subterranean Centrosaurus bone core (surface scraped prior to powdering), BEDTA = matrix surrounded subterranean Centrosaurus bone core (EDTA demineralised, surface scraped prior to powdering), M = adjacent mudstone matrix of subterranean Centrosaurus bone, S = Surface scrapings from matrix-surrounded subterranean Centrosaurus bone. The dark green bands are sequences phylogenetically close to Euzebya. See the supplemental files for a full listing of taxa.

https://doi.org/10.7554/eLife.46205.100
Appendix 1—figure 59
Comparison of microbial community (phylum level) from matrix-surrounded subterranean.

Centrosaurus bone, bone scrapings, and adjacent mudstone matrix. There are two replicates per sample. Sample ID’s are as in Appendix 1—table 2: B = matrix surrounded subterranean Centrosaurus bone core (surface scraped prior to powdering), BEDTA = matrix surrounded subterranean Centrosaurus bone core (EDTA demineralised, surface scraped prior to powdering), M = adjacent mudstone matrix of subterranean Centrosaurus bone, S = Surface scrapings from matrix-surrounded subterranean Centrosaurus bone.

https://doi.org/10.7554/eLife.46205.101
Appendix 1—figure 60
PCA of species-level 16S rRNA amplicon sequence data (percentages without additional normalization).

Red triangles with one vertex upward are from the bone core, red triangles with one vertex downward are the EDTA demineralized bone core, yellow circles are the bone surface scrapings, and brown circles are the mudstone. PC1 and PC2 account for 75.87% and 21.65% of the variation in the data, respectively.

https://doi.org/10.7554/eLife.46205.102

Tables

Table 1
Comparison of Late Cretaceous, Pleistocene-Holocene, and modern amino acid racemization values of the KOH-treated samples.

NA indicates that amino acid concentration was below detection limit.

https://doi.org/10.7554/eLife.46205.010
Sample treatmentAsx D/LGlx D/LSer D/LAla D/LVal D/L
Matrix-surrounded subterranean Centrosaurus bone
Ethanol rinsed before powdering, gelatedNANANANANA
Subterranean Centrosaurus bone uncovered from matrix before collection
Ethanol rinsed before powdering, gelated0.210.5500.210
Adjacent mudstone matrix of subterranean Centrosaurus bone
Ethanol rinsed before powdering, gelatedNANA00.300
Surface-eroded Centrosaurus bone from BB180
Ethanol rinsed before powdering, gelated00000
Surface-eroded Late Cretaceous bone on same ridge and ~ 21 m above BB180
Ethanol rinsed before powdering, gelated00.9500.320.90
Topsoil on same ridge and ~ 64 m above BB180
Ethanol rinsed before powdering, gelated0.140.140.050.090.04
Pleistocene-Holocene surface-eroded Carcharias teeth
Unrinsed0.210.040.090.030.01
Ethanol rinsed before powdering0.510.150.300.160.11
Ethanol rinsed before powdering0.530.150.300.170.11
Modern Gallus bone
Unrinsed0.050.0300.020
Ethanol rinsed before powdering0.060.0300.020
Table 2
Carbon data from Late Cretaceous fossil bone, mudstone, topsoil, and younger bone.
https://doi.org/10.7554/eLife.46205.011
Sample% mass after HCl demineralizationC % (organic fraction)F14C (organic fraction)
Matrix-surrounded subterranean Centrosaurus bone core (surface scraped prior to powdering)53.982.7770.0149
Adjacent mudstone matrix of subterranean Centrosaurus bone82.271.320.0573
Topsoil on same ridge and ~ 64 m above BB18091.6320.766
Mudstone on same ridge and ~ 23 m above BB18090.380.890.0628
Surface-eroded Late Cretaceous bone core on same ridge and ~ 21 m above BB180 (surface scraped prior to powdering)43.41.630.0422
Yarnton bovine right femur (82–71 ka, Cook et al., 2012)16.7344.90.0056*
  1. *This sample was used for blank correction in the AMS analyses, therefore this value is not blank-subtracted.

Table 3
DNA concentrations in mudstone matrix and bone quantified with Qubit fluorometry.
https://doi.org/10.7554/eLife.46205.013
SampleAverage DNA concentration (ng/μL)Total DNA (ng)DNA per 1 g of bone or mudstone (ng/g)
Matrix-surrounded subterranean
Centrosaurus bone core
(surface scraped prior to powdering)
0.793965793
Adjacent mudstone matrix of
subterranean Centrosaurus bone
0.0316416.4
Laboratory blankBelow detection (<0.01*)
  1. *Note: the detection limit corresponds to the actual concentration of DNA in the assay tube (0.0005 ng/µL) after 200 times dilution of the original sample according to the manufacturer’s protocol.

Appendix 1—table 1
Average human body composition and hydrolysis calculations.
https://doi.org/10.7554/eLife.46205.017
Substance% of body massAverage adult body mass (kg)Mass of substance (kg)Mass of substance (Da)Typical amino acid (Da)Number of amino acidsMaximum number of peptide bondsNumber of bonds surviving
Protein206212.47.46728E + 271106.78844E + 256.78844E + 25−1.25963E + 27
H2O (Da)Number of H2O molecules% H2O molecules used up in hydrolysis
Water646239.682.38953E + 28181.32752E + 275.113636364
Appendix 1—table 2
Sample ID key and descriptions.
https://doi.org/10.7554/eLife.46205.025
Sample bag #Originally sent toHplc idPy-GC/MS IDDNA extraction, fluorescence microscopy, 16S rRNA amplicon IDLocationTypeDetails
1 (TMP 2016.016.0007)PrincetonNANA1B, 1S, 1FDinosaur Provincial Park, Alberta, CanadaBoneCentrosaurus rib aseptically collected within immediately surrounding sediment. One end of the rib was first exposed. Aseptic protocol was then implemented to expose more of the rib. A rib section was isolated with the surrounding sediment kept in situ. Foil was placed on top of this section. The bone was sawed on its ends and then flipped by prying underneath with an awl. Mudstone tended to fracture during flipping. More foil was added after flipping to encase the whole sample.
1 (TMP 2016.016.0007)PrincetonNANA1MDinosaur Provincial Park, Alberta, CanadaMudstone matrixMudstone matrix surrounding and collected with the matrix-surrounded Centrosaurus rib sample. Had tendency to fracture when manipulated.
2 (TMP 2016.016.0007)PrincetonNANA2B, 2SDinosaur Provincial Park, Alberta, CanadaBoneUncovered Centrosaurus rib section immediately adjacent to matrix-surrounded section of sample bag #1.
6 (TMP 2016.016.0013)PrincetonNANA6B, 6SDinosaur Provincial Park, Alberta, CanadaBoneSurface bone eroded out of BB180, either excavated in past years and left or naturally eroded. About eight steps away from quarry cliff-face. 667 m elevation.
8 (TMP 2016.016.0014)PrincetonNANANADinosaur Provincial Park, Alberta, CanadaMudstone sedimentSediment from BB180 at bone producing layer. Sampled ~ 30 cm away from the sampled rib and tibia (sample bags #1–4). 670 m elevation.
10 (TMP 2016.016.0015)PrincetonNANA10, 10TDinosaur Provincial Park, Alberta, CanadaTopsoilTopsoil from same ridge as BB180. 734 m elevation.
11 (TMP 2016.016.0016)PrincetonNANANADinosaur Provincial Park, Alberta, CanadaMudstone sedimentSediment on same ridge as BB180 from 709 m elevation. Outcrop was dug into by several cm before sampling.
13 (TMP 2016.016.0017)PrincetonNANA13Dinosaur Provincial Park, Alberta, CanadaMudstone sedimentSediment on same ridge as BB180 from 693 m elevation. Outcrop was dug into by several cm before sampling.
16 (TMP 2016.016.0018)PrincetonNANA16B, 16SDinosaur Provincial Park, Alberta, CanadaBoneSurface bone eroded out of same ridge as BB180 but at 691 m elevation. Unknown taxon. Near sample bags #13–14.
NABristol11NASainsbury’s Bristol, UKBoneChicken bone purchased from grocery store with meat removed.
3 (TMP 2016.016.0008)Bristol22NADinosaur Provincial Park, Alberta, CanadaBoneCentrosaurus tibia aseptically collected within immediately surrounding sediment. One end of the tibia was first exposed. Aseptic protocol was then implemented to expose more of the tibia. A tibia section was isolated with the surrounding sediment kept in situ. Foil was placed on top of this section. The bone was sawed on its ends and then flipped by prying underneath with an awl. Mudstone tended to fracture during flipping. More foil was added after flipping to encase the whole sample.
3 (TMP 2016.016.0008)Bristol33NADinosaur Provincial Park, Alberta, CanadaMudstone matrixMudstone matrix surrounding and collected with the matrix-surrounded Centrosaurus tibia. Had tendency to fracture when manipulated.
4 (TMP 2016.016.0008)Bristol44NADinosaur Provincial Park, Alberta, CanadaBoneUncovered Centrosaurus tibia region immediately adjacent to matrix-surrounded section of sample bag #3.
5 (TMP 2016.016.0013)Bristol66NADinosaur Provincial Park, Alberta, CanadaBoneSurface bone eroded out of BB180, either excavated in past years and left or naturally eroded. About eight steps away from quarry cliff-face. 667 m elevation.
7 (TMP 2016.016.0014)Bristol55NADinosaur Provincial Park, Alberta, CanadaMudstone sedimentSediment from BB180 at bone producing layer. Sampled ~ 30 cm away from the sampled rib and tibia (sample bags #1–4). 670 m elevation.
9 (TMP 2016.016.0015)Bristol77NADinosaur Provincial Park, Alberta, CanadaTopsoilTopsoil from same ridge as BB180. 734 m elevation.
12 (TMP 2016.016.0016)Bristol88NADinosaur Provincial Park, Alberta, CanadaMudstone sedimentSediment on same ridge as BB180 from 709 m elevation. Outcrop was dug into by several cm before sampling.
14 (TMP 2016.016.0017)Bristol99NADinosaur Provincial Park, Alberta, CanadaSedimentSediment on same ridge as BB180 from 693 m elevation. Outcrop was dug into by several cm before sampling.
15 (TMP 2016.016.0018)Bristol1010NADinosaur Provincial Park, Alberta, CanadaBoneSurface bone eroded out of same ridge as BB180 but at 691 m elevation. Unknown taxon. Near sample bags #13–14.
Appendix 1—table 3
Pilot samples of fossil Mesozoic bone THAA composition.

Concentrations in picomoles/mg.

https://doi.org/10.7554/eLife.46205.039
TaxonDetailsLocationApproxi
mate
age
Notes[Asx][Glx][Ser][L-Thr][L-His][Gly][L-Arg][Ala][Tyr][Val][Phe][Leu][Ile][Total]
hadrosaur?unrinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceoussignal suppression81126153081.61433241191.030656101174
hadrosaur?unrinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceoussignal suppression140175230670.0056801082.05276001417
hadrosaur?unrinsed, interior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceoussignal suppression5797135280.0043230733.0263800919
hadrosaur?unrinsed, interior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceoussignal suppression58102123310.0035430844.030323123902
hadrosaur?gelated, unrinsed, interior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousweaker signal suppression4684104250.00356257212.023392718831
sauropod?unrinsed,, high in siderite, Morrison Fmnear Grass Range, Montana, USALate Jurassicstrong signal suppression0127000.00482014913.00000772
hadrosaur?ethanol rinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousstrong signal suppression8411916400.004659506.005900991
hadrosaur?ethanol rinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousstrong signal suppression68105145390.003480767.00000789
hadrosaur?gelated. ethanol rinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousweaker signal suppression5693132350.0029406214.037403221816
hadrosaur?ethanol rinsed, interior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceoussignal suppression60116174300.003150648.0283800832
hadrosaur?ethanol rinsed, interior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceoussignal suppression571041643873.672860569.028333016895
hadrosaur?gelated, ethanol rinsed, interior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousweaker signal suppression50981493242.43280135815.026393018848
sauropod?gelated, ethanol rinsed, high in siderite, Morrison Fmnear Grass Range, Montana, USALate Jurassicstill strong signal suppression0982411290.009970016.000001481
hadrosaur?unrinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousno signal suppression93710.002111212.0641582
hadrosaur?unrinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousno signal suppression844085.983711513.06544152
Alioramusloaned from American Museum of Natural HistoryMongoliaLate Cretaceouspoor amino acid profile00000.0000017.0000017
Alioramusethanol rinsed, loaned from American Museum of Natural HistoryMongoliaLate Cretaceousdecent amino acid profile29408900.009806119.20000336
Alioramusgelated, ethanol rinsed, loaned from American Museum of Natural HistoryMongoliaLate Cretaceouspoor amino acid profile00000.0000021.4000021
Appendix 1—table 4
Pilot samples of fossil Mesozoic bone THAA D/L values.
https://doi.org/10.7554/eLife.46205.040
TaxonDetailsLocationApproxi
mate
age
NotesAsx D/LGlx D/LSer D/LArg D/LAla D/LVal D/LPhe D/LLeu D/LIle D/LTyr D/L
hadrosaur?unrinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceoussignal suppression0.1850.1210.0000.0000.5520.0000.0001.071NANA
hadrosaur?unrinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceoussignal suppression0.1850.0000.000NA0.0000.0000.000NANANA
hadrosaur?unrinsed, interior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceoussignal suppression0.0000.0000.0000.0000.0000.0000.000NANA0.000
hadrosaur?unrinsed, interior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceoussignal suppression0.0000.0000.0000.0000.0000.0000.0000.0000.000NA
hadrosaur?gelated, unrinsed, interior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousweaker signal suppression0.0000.0000.0000.0000.0000.0000.0000.0000.000NA
sauropod?unrinsed, high in siderite, Morrison Fmnear Grass Range, Montana, USALate Jurassicstrong signal suppressionNA0.000NANA0.000NANANANANA
hadrosaur?ethanol rinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousstrong signal suppression0.0000.0000.0000.000NANA0.000NANA0.000
hadrosaur?ethanol rinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousstrong signal suppression0.0000.0000.000NA0.000NANANANANA
hadrosaur?gelated, ethanol rinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousweaker signal suppression0.0000.0000.000NA0.0000.0000.0000.0000.000NA
hadrosaur?ethanol rinsed, interior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceoussignal suppression0.0000.0000.000NA0.0000.0000.000NANA0.000
hadrosaur?ethanol rinsed, interior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceoussignal suppression0.0000.0000.000NA0.0000.0000.0000.0000.000NA
hadrosaur?gelated, ethanol rinsed, interior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousweaker signal suppression0.0000.0000.0001.8420.0000.0000.0000.0000.000NA
sauropod?gelated, ethanol rinsed, high in siderite, Morrison Fmnear Grass Range, Montana, USALate Jurassicstill strong signal suppressionNA0.0000.000NANANANANANANA
hadrosaur?unrinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousno signal suppression0.0000.4260.8950.0000.0000.0000.4280.0000.0000.000
hadrosaur?unrinsed, exterior, Dinosaur Park FmDinosaur Provincial Park, Alberta, CanadaLate Cretaceousno signal suppression0.0840.1630.0141.5240.0890.1080.2450.0000.0000.134
Alioramusloaned from American Museum of Natural HistoryMongoliaLate Cretaceouspoor amino acid profileNANANANANANANANANANA
Alioramusethanol rinsed, loaned from American Museum of Natural HistoryMongoliaLate Cretaceousdecent amino acid profile0.0000.0000.000NA0.000NANANANANA
Alioramusgelated, ethanol rinsed, loaned from American Museum of Natural HistoryMongoliaLate Cretaceouspoor amino acid profileNANANANANANANANANANA
Appendix 1—table 5
Shark teeth THAA composition.

Concentrations in picomoles/mg.

https://doi.org/10.7554/eLife.46205.041
SpeciesDetailsLocationApprox
imate
age
Notes[Asx][Glx][Ser][L-Thr][L-His][Gly][L-Arg][Ala][Tyr][Val][Phe][Leu][Ile][Total]
Carcharias taurusunrinsed, unnamed Pleistocene-Holocene sedimentsPonte Vedra Beach, Florida, USAQuaternaryhigh
concentration
526565333729149170644643.2526616031884764815.01657793662046219071596855
Carcharias taurusethanol rinsed, unnamed Pleistocene-Holocene sedimentsPonte Vedra Beach, Florida, USAQuaternaryweak
signal suppression
4784711046558.31230918843010.0194932291954824
Carcharias taurusethanol rinsed, unnamed Pleistocene-Holocene sedimentsPonte Vedra Beach, Florida, USAQuaternaryweak
signal suppression
5885341157361.79261720353711.02611202722555647
Appendix 1—table 6
Shark teeth THAA D/L values.
https://doi.org/10.7554/eLife.46205.042
SpeciesDetailsLocationApproximate
age
NotesAsx D/LGlx D/LSer D/LArg D/LAla D/LVal D/LPhe D/LLeu D/LIle D/LTyr D/L
Carcharias
taurus
unrinsed,
unnamed
Pleistocene-
Holocene
sediments
Ponte Vedra
Beach, Florida,
USA
Quaternaryhigh
concen
tration
0.2090.0390.0920.0470.0270.0110.0280.0260.1650.114
Carcharias
taurus
ethanol
rinsed,
unnamed
Pleistocene-
Holocene
sediments
Ponte Vedra
Beach, Florida,
USA
Quaternaryweak
signal
suppression
0.5120.1530.3010.2360.1550.1140.0000.0920.1070.000
Carcharias
taurus
ethanol
rinsed,
unnamed
Pleistocene-
Holocene
sediments
Ponte Vedra
Beach, Florida,
USA
Quaternaryweak signal
suppression
0.5270.1540.2950.3580.1660.1120.1580.0940.106NA
Appendix 1—table 7
Aseptically collected samples and chicken control THAA composition.

See Appendix 1—table 2 for Sample ID’s. e = ethanol rinsed before powdering. d = gelated. Concentrations in picomoles/mg.

https://doi.org/10.7554/eLife.46205.043
Sample ID[Asx][Glx][Ser][L-Thr][L-His][Gly][L-Arg][Ala][Tyr][Val][Phe][Leu][Ile][Total]
171588105116455643332717233.813854025805713667114.7383802639757808248291000385
1e81260124003510603654722168.254459666626115721617.4420572871456845271661139278
200000.002090014.80000224
2041000.001820015.00000239
2e5660000.0018106817.60000383
2ed00000.00330019.8000052
3182056350.0024509715.238216460787
3353299450.00190011715.4491054533765
3e002000.0011107617.80000224
3ed0024170.009207520.0280037294
429324300.0015804115.604300360
4e2342271771080.00509022018.01480001642
4ed8711051450.00191179520.257295336791
500000.0000015.8000016
5e064000.001840018.10000266
5ed154016150.007903320.3160190255
6104131100910.005336616415.97792102501526
6148144116970.003185216316.18959104551361
6e36752830528466.1572317750318.32531603321623878
6ed578968630.001773810620.564327238825
793264315161157904.345438898252816.31153675122665117737
7e3541306834673424799.16115682024637718.5350818083972206145636
7ed3505271716741818178.644659541312520.719479002035114824268
83933062622820.0083414251616.53231754152173880
8e00000.0000018.7000019
8ed9111570.002401720.99000114
900000.0000016.7000017
9e003600.00003418.9000089
9ed18484500.008203521.10000249
10597747400.001853810416.9540680688
10e39199000.0014807019.113901690782
10ed15862300.007804321.369156545462
Appendix 1—table 8
Aseptically collected samples and chicken control THAA D/L values.

See Appendix 1—table 2 for Sample ID’s. e = ethanol rinsed before powdering. d = gelated.

https://doi.org/10.7554/eLife.46205.044
Sample IDAsx D/LGlx D/LSer D/LArg D/LAla D/LVal D/LPhe D/LLeu D/LIle D/LTyr D/L
10.0530.0270.0000.0960.0150.0000.0190.0220.0000.000
1e0.0550.0290.0000.0810.0160.0000.0000.0000.0000.000
2NANANANANANANANANANA
2NA0.000NANANANANANANANA
2e0.0000.000NANA0.000NANANANANA
2edNANANANANANANANANANA
30.0000.0000.000NA0.0000.0000.0000.000NANA
30.0000.0000.000NA0.0000.0000.0000.0000.000NA
3eNANA0.000NA0.338NANANANANA
3edNANA0.000NA0.2990.000NANA0.000NA
40.0000.0000.000NA0.000NA0.000NANANA
4e0.0000.0000.000NA0.0000.000NANANANA
4ed0.2140.5500.0000.9890.2070.0000.0000.0000.000NA
5NANANANANANANANANANA
5eNA0.000NANANANANANANANA
5ed0.0000.7270.000NA0.0000.000NA0.000NANA
60.0000.0000.0001.9960.0000.0000.0000.0000.000NA
60.1100.0000.0001.6450.0000.0000.0000.0000.000NA
6e0.0820.0840.0000.8890.0780.0000.0000.0000.000NA
6ed0.0000.0000.0001.3380.0000.0000.0000.0000.000NA
70.4180.3780.0200.8870.0710.0000.0000.0560.0000.000
7e0.1300.1230.0380.8560.0800.0380.0480.0700.0000.000
7ed0.1350.1390.0450.6390.0890.0400.0490.0630.000NA
80.1250.0000.0001.1480.0000.0000.0000.0000.0000.000
8eNANANANANANANANANANA
8ed0.0000.0000.000NA0.0000.000NANANANA
9NANANANANANANANANANA
9eNANA0.000NA0.000NANANANANA
9ed0.0001.0550.000NA0.000NANANANANA
100.0000.0000.0000.0000.0000.000NA0.000NANA
10e0.0000.538NANA0.0000.750NA0.675NANA
10ed0.0000.9510.000NA0.3230.9010.0000.7510.677NA
Appendix 1—table 9
Summary of variable loadings for the non-normalised PCA of THAA composition of only the sufficiently treated samples associated with Figure 6F.

Proportion of variance for each principal component in parentheses.

https://doi.org/10.7554/eLife.46205.046
Pc1 (55.04%)Pc2 (22.66%)
Asx−0.100509897−0.18614726
Glx−0.214376490−0.05665852
Ser−0.1442865120.18384010
L. Thr−0.0988054310.04356834
L. His0.007992613−0.05584073
Gly0.805294439−0.41882274
L. Arg0.009652324−0.20786057
Alo−0.185328976−0.04346374
Tyr0.4018271500.82375170
Val−0.1860190660.10343138
Phe−0.048918108−0.06420351
Leu−0.150726139−0.09272073
Ile−0.095795908−0.02887370
Appendix 1—table 10
Summary of variable loadings for the normalised PCA of THAA composition of only the sufficiently treated samples associated with Appendix 1—figure 22.

Proportion of variance for each principal component in parentheses.

https://doi.org/10.7554/eLife.46205.049
Pc1 (31.64%)Pc2 (25.42%)
Asx−0.24729982−0.247493801
Glx−0.259250170.007025074
Ser−0.086723070.347552010
L. Thr−0.252821040.153203703
L. His−0.02073369−0.470955522
Gly0.40229882−0.284333925
L. Arg−0.09443713−0.475273301
Alo−0.200198390.215195078
Tyr0.370120330.243369947
Val−0.353723960.243119598
Phe−0.36466490−0.264010157
Leu−0.36975651−0.118102708
Ile−0.238978480.132700762
Appendix 1—table 11
Summary of variable loadings for the normalised PCA of THAA composition associated with the plot in Appendix 1—figure 24F.

Proportion of variance for each principal component in parentheses.

https://doi.org/10.7554/eLife.46205.052
Pc1 (30.01%)Pc2 (15.97%)
Asx−0.33562690.10557119
Glx−0.187365320.40091324
Ser−0.07675319−0.57396909
L. Thr−0.38235418−0.13868623
L. His−0.197024910.04051298
Gly0.110684340.37647168
L. Arg−0.311472850.09964743
Alo−0.17744465−0.50906983
Tyr0.35387327−0.09153454
Val−0.390699290.05903456
Phe−0.11192988−0.14530964
Leu−0.375684450.16792075
Ile−0.29841571−0.07628272
Appendix 1—table 12
Summary of variable loadings for the non-normalised PCA of THAA composition associated with Appendix 1—figure 26.

Proportion of variance for each principal component in parentheses.

https://doi.org/10.7554/eLife.46205.055
Pc1 (62.09%)Pc2 (24.49%)
Asx−0.0613341260.101860246
Glx−0.1016895120.042538223
Ser−0.0175847750.227710833
L. Thr−0.0267882910.091854076
L. His−0.0063343890.006290775
Gly−0.438716137−0.826790362
L. Arg−0.0243602710.030568893
Alo−0.0797622510.26975096
Tyr0.883822618−0.350122096
Val−0.0357506180.142845467
Phe−0.0298040140.062677703
Leu−0.0406209280.139913899
Ile−0.0210773070.060901383
Appendix 1—table 13
Comparison of Late Cretaceous, Pleistocene-Holocene, and modern amino acid racemisation values.

NA indicates that amino acid concentration was below detection limit.

https://doi.org/10.7554/eLife.46205.057
Sample treatmentAsx D/LGlx D/LSer D/LAla D/LVal D/L
Matrix-surrounded subterranean Centrosaurus bone
UnrinsedNANANANANA
UnrinsedNA0NANANA
Ethanol rinsed before powdering00NA0NA
Ethanol rinsed before powdering, gelatedNANANANANA
Subterranean Centrosaurus bone uncovered from matrix before collection
Unrinsed0000NA
Ethanol rinsed before powdering00000
Ethanol rinsed before powdering, gelated0.2140.55000.2070
Adjacent mudstone matrix of subterranean Centrosaurus bone
Unrinsed00000
Unrinsed00000
Ethanol rinsed before powderingNANA00.338NA
Ethanol rinsed before powdering, gelatedNANA00.2990
Surface-eroded Centrosaurus bone from BB180
Unrinsed00000
Unrinsed0.1100000
Ethanol rinsed before powdering0.0820.08400.0780
Ethanol rinsed before powdering, gelated00000
Surface-eroded Late Cretaceous bone on same ridge and ~ 21 m above BB180
Unrinsed00000
Ethanol rinsed before powdering00.538NA00.750
Ethanol rinsed before powdering, gelated00.95100.3230.901
Topsoil on same ridge and ~ 64 m above BB180
Unrinsed0.4180.3780.0200.0710
Ethanol rinsed before powdering0.1300.1230.0380.0800.038
Ethanol rinsed before powdering, gelated0.1350.1390.0450.0890.040
Pleistocene-Holocene surface-eroded Carcharias teeth
Unrinsed0.2090.0390.0920.0270.011
Ethanol rinsed before powdering0.5120.1530.3010.1550.114
Ethanol rinsed before powdering0.5270.1540.2950.1660.112
Modern Gallus bone
Unrinsed0.0530.02700.0150
Ethanol rinsed before powdering0.0550.02900.0160
Appendix 1—table 14
EDS eZAF Smart Quant results of demineralised modern chicken bone full area 1.
https://doi.org/10.7554/eLife.46205.069
ElementWeight %Atomic %Net int.Error %KratioZRAF
C K21.8827.49191.178.980.06661.04130.98030.29211.0000
N K24.8526.76138.3610.570.04511.01690.99060.17861.0000
O K44.1741.65471.579.960.06790.99570.99990.15431.0000
AlK1.770.99134.207.250.01030.88461.03630.65451.0066
SiK0.610.3354.639.510.00430.90381.04220.76581.0100
P K0.210.1016.9323.700.00160.86791.04770.85761.0160
S K1.530.72132.124.250.01270.88481.05290.92371.0199
ClK4.101.74322.092.470.03340.84161.05780.95531.0138
FeK0.240.077.4755.870.00220.75931.08621.02121.1884
CuK0.630.1512.8530.910.00610.72641.08581.01751.2983
Appendix 1—table 15
EDS eZAF Smart Quant results of demineralised Pleistocene-Holocene shark tooth full area 1.
https://doi.org/10.7554/eLife.46205.072
ElementWeight %Atomic %Net int.Error %KratioZRAF
C K25.6542.51150.1610.580.04011.10740.93670.14111.0000
O K27.7934.58542.959.500.05971.06200.95920.20241.0000
AlK0.390.2935.9712.820.00210.94791.00180.55871.0102
SiK0.880.6299.397.890.00600.96911.00880.69011.0169
S K21.6313.432464.072.580.18070.94991.02170.86951.0112
ClK0.760.4368.5910.210.00540.90391.02770.77371.0166
FeK22.458.00847.322.190.19270.81891.06581.00391.0442
CuK0.440.1410.2441.090.00370.78471.06920.98491.0840
Appendix 1—table 16
EDS eZAF Smart Quant results of mineral grain from demineralised Late Cretaceous Centrosaurus bone area A.
https://doi.org/10.7554/eLife.46205.075
ElementWeight %Atomic %Net int.Error %KratioZRAF
C K8.6814.27101.9399.990.01071.08740.95450.11331.0000
O K40.7050.272568.998.500.11131.04150.97590.26251.0000
AlK1.060.78326.725.090.00750.92741.01630.74181.0360
SiK48.4634.0916392.382.650.38720.94791.02290.84101.0025
S K0.400.2580.679.760.00230.92841.03500.62791.0056
ClK0.010.012.3160.700.00010.88321.04050.72591.0084
CaK0.690.34125.278.440.00570.89501.05520.91031.0202
Appendix 1—table 17
EDS eZAF Smart Quant results of vessel (spot 1) from demineralised Late Cretaceous Centrosaurus bone area B.
https://doi.org/10.7554/eLife.46205.078
ElementWeight %Atomic %Net int.Error %KratioZRAF
C K5.619.6681.0599.990.00711.10050.94640.11521.0000
O K40.9552.943343.328.290.12121.05460.96830.28071.0000
AlK3.752.881306.454.480.02530.94001.00970.69871.0256
SiK44.5132.7816780.973.330.33200.96091.01650.77391.0031
S K0.870.56212.947.260.00520.94141.02900.62481.0067
CaK0.960.50211.926.870.00810.90791.05010.90591.0253
BaL2.460.37177.238.150.01840.65371.25261.08221.0599
FeK0.400.1546.6713.640.00350.81001.07080.99441.0873
CuK0.490.1636.9317.790.00440.77561.07331.00441.1609
Appendix 1—table 18
EDS eZAF Smart Quant results of fibrous mass (spot 2) from demineralised Late Cretaceous Centrosaurus bone area B.
https://doi.org/10.7554/eLife.46205.080
ElementWeight %Atomic %Net int.Error %KratioZRAF
C K5.9410.3862.7399.990.00701.10210.94640.10761.0000
O K36.5347.942200.108.490.10211.05620.96840.26461.0000
AlK5.474.261553.474.170.03850.94131.00980.72761.0272
SiK48.1636.0114261.523.300.36110.96221.01660.77711.0027
S K0.570.37105.798.710.00330.94261.02910.60741.0061
K K0.060.0312.0555.960.00050.89271.04530.85361.0188
CaK0.790.42135.546.590.00660.90911.05010.89851.0229
BaL1.430.2280.4112.010.01070.65461.25271.07841.0639
FeK0.340.1331.1416.610.00300.81091.07080.99511.0943
CuK0.710.2442.6216.370.00650.77651.07331.00491.1673
Appendix 1—table 19
EDS eZAF Smart Quant results of vessel from demineralised Late Cretaceous Centrosaurus bone area C.
https://doi.org/10.7554/eLife.46205.083
ElementWeight %Atomic %Net int.Error %KratioZRAF
C K7.7813.4585.9099.990.01001.10290.94360.11601.0000
O K37.9449.222221.658.490.10641.05720.96570.26531.0000
AlK1.561.20407.625.190.01040.94261.00740.69111.0273
SiK46.3034.2213551.973.160.35430.96361.01430.79131.0032
S K0.840.54154.407.870.00490.94421.02690.62171.0071
CaK1.010.52169.406.800.00860.91081.04820.90411.0273
BaL3.730.56203.067.220.02790.65591.25071.08061.0545
FeK0.410.1535.8517.080.00360.81281.06940.99121.0797
CuK0.430.1424.3821.060.00380.77841.07211.00271.1492
Appendix 1—table 20
EDS eZAF Smart Quant results of vessel exterior (region 1) from demineralised Late Cretaceous Centrosaurus bone area D.
https://doi.org/10.7554/eLife.46205.086
ElementWeight %Atomic %Net int.Error %KratioZRAF
C K10.9917.49159.5099.990.01481.08320.95650.12451.0000
O K43.5552.053194.438.390.12241.03740.97780.27081.0000
AlK1.621.15541.464.850.01100.92371.01780.71661.0296
SiK42.3728.8515672.002.920.32650.94401.02440.81421.0026
S K0.160.1038.7515.680.00100.92461.03640.64961.0060
CaK0.310.1565.749.960.00260.89141.05640.92111.0232
BaL0.530.0736.7427.530.00400.64161.25931.09541.0750
FeK0.170.0619.3724.430.00150.79471.07561.00311.1088
CuK0.290.0921.5321.860.00270.76071.07731.00901.2037
Appendix 1—table 21
EDS eZAF Smart Quant results of vessel interior (region 2) from demineralised Late Cretaceous Centrosaurus bone area D.
https://doi.org/10.7554/eLife.46205.088
ElementWeight %Atomic %Net int.Error %KratioZRAF
C K25.0935.59183.4499.990.04671.06600.96500.17471.0000
O K43.4946.31999.608.890.10491.02040.98570.23651.0000
AlK4.182.64475.034.960.02650.90801.02460.68781.0165
SiK24.2514.713016.293.570.17230.92791.03090.76301.0034
S K0.170.0916.8423.850.00120.90871.04240.73681.0077
CaK0.430.1833.6913.240.00370.87601.06160.95721.0302
BaL1.180.1529.8927.690.00900.63051.26471.11721.0818
FeK0.190.067.8755.090.00170.78091.07961.00941.1220
CuK1.010.2726.6317.380.00910.74731.08051.01201.1953
Appendix 1—table 22
EDS eZAF Smart Quant results of mineral grain from demineralised Late Cretaceous Centrosaurus bone area E, spot 1.
https://doi.org/10.7554/eLife.46205.091
ElementWeight %Atomic %Net int.Error %KratioZRAF
C K8.1219.8576.3599.990.01811.23240.86230.18131.0000
O K21.3239.16732.398.550.07181.18570.88690.28381.0000
AlK4.945.38431.027.600.02251.06490.93560.42371.0087
SiK21.7422.742311.266.070.12331.08990.94400.51691.0068
S K1.451.33146.928.190.00961.07050.95970.60861.0162
K K0.270.2027.8218.410.00241.01740.98100.83631.0548
CaK2.621.92249.094.580.02571.03730.98750.88141.0698
BaL36.017.701044.052.700.29290.74951.18541.05261.0312
FeK1.340.7159.0510.930.01200.93251.02020.91451.0442
CuK2.191.0163.317.260.02030.89761.03060.95911.0793
Appendix 1—table 23
Non-demineralised Dinosaur Provincial Park samples.
https://doi.org/10.7554/eLife.46205.092
SampleMass analysed (mg)C %
Matrix-surrounded subterranean bone (not scrapped)3.2192.3
Adjacent mudstone matrix of subterranean bone2.1121.11
BB180 surface bone (not scrapped)3.1844.19
Topsoil2.2351.26
Mudstone 693 m elevation3.4131.15
Appendix 1—table 24
Pilot tests on demineralised Dinosaur Provincial Park samples (0.5 M HCl).
https://doi.org/10.7554/eLife.46205.093
SampleMass (mg) bulk powder before demineralisationMass (mg) after demineralisation% mass surviving demineralisationMass (mg) analysedC %% change in [C] from bulk powder
Matrix-surrounded subterranean bone (not scrapped)299.77.5362.5145145154.03513.48486.0869565
Adjacent mudstone matrix of subterranean bone308.5253.882.269043763.4991.2815.31531532
BB180 surface bone (not scrapped)307.171.9923.4418756110.0510.13−96.8973747
Topsoil307.2281.591.634114586.211.4313.49206349
Mudstone 693 m elevation300.4271.590.379494015.0050.92−20
Surface bone 691 m elevation (core)34114843.401759537.9031.47NA
Appendix 1—table 25
Quebit fluorometer test results on Dinosaur Provincial Park samples.

Sample ID’s as in Appendix 1—table 2.

https://doi.org/10.7554/eLife.46205.094
Bag
number
TypeReplicateSample
ID
KitNg of DNA / μLConcentrationConcentrated ng
of DNA / μL
First readSecond readFirst readSecond read
NABlankNABlankPower
Viral
Below
detection
Below
detection
10Topsoil110Power
Viral
0.1510.133
13Mudstone113Power
Viral
Below
detection
Below
detection
16Scrappings116SPower
Viral
0.1720.164
16Bone116B1Power
Viral
0.4240.404
16Bone216B2Power
Viral
0.5920.55
NABlankNABlankPower
Viral
Below
detection
Below
detection
1Float11F1Power
Viral
0.09260.0908
1Scrappings11S1Power
Viral
0.1280.127
1Scrappings21S2Power
Viral
0.02380.0236
1Bone11B1Power
Viral
0.03820.0376
1Bone21B2Power
Viral
0.05440.0546
1Mudstone11M1Power
Viral
Below
detection
Below
detection
1Mudstone21M2Power
Viral
Below
detection
Below
detection
NABlankNABlankDneasy
PowerMax
Soil
Below
detection
Below
detection
x 25Below
detection
Below
detection
1Bone31B5gDneasy
PowerMax
Soil
0.7980.788x 2511.110.5
1Mudstone31M10gDneasy
PowerMax
Soil
0.03340.0322x 250.6260.612
1Mudstone41M2Dneasy
PowerMax
Soil
0.06240.0596x 250.5860.586
8Mudstone18M1Dneasy
PowerMax
Soil
0.0960.0924x 251.641.58
8Mudstone28M2Dneasy
PowerMax
Soil
0.1440.141x 251.61.55
11Mudstone111M1Dneasy
PowerMax
Soil
Below
detection
Below
detection
x 250.03060.0292
11Mudstone211M2Dneasy
PowerMax
Soil
Below
detection
Below
detection
x 250.02180.021
13Mudstone213M1Dneasy
PowerMax
Soil
Below
detection
Below
detection
x 250.1230.119
13Mudstone313M2Dneasy
PowerMax
Soil
0.01660.016x 250.1410.138
2Scrappings12S1Power
Viral
0.1670.168
2Scrappings22S2Power
Viral
0.1130.109
2Bone12B1Power
Viral
0.1340.131
2Bone22B2Power
Viral
0.1180.116
6Scrappings16S1Power
Viral
Below
detection
Below
detection
6Scrappings26S2Power
Viral
0.0114Below
detection
6Bone16B1Power
Viral
Below
detection
Below
detection
6Bone26B2Power
Viral
0.0102Below
detection
1Float21F2Power
Viral
0.0290.0278
10Topsoil210T2Power
Viral
1.041.02
6Scrappings36S3Power
Viral
x 20.2080.206
6Bone36B3Power
Viral
x 20.04220.0424
1Bone
(EDTA
demineralised)
11BEDTADneasy
PowerMax
Soil
0.1480.145x 203.523.44
6Bone
(EDTA
demineralised)
16BEDTADneasy
PowerMax
Soil
0.01440.013x 200.3240.318
Appendix 1—table 26
Cell abundance calculations for dinosaur bone and adjacent mudstone matrix from amino acid and DNA abundance based on Lomstein et al. (2012), Onstott et al. (2014), and Magnabosco et al. (2018).
https://doi.org/10.7554/eLife.46205.095
Amino acids
BoneMudstone
picomoles/mg50300picomoles/mg
nanomoles/g of bone50300nanomoles/g of mudstone
g/mole117.4113.8g/mole
grams of AA/g8.39E-064.88E-05grams of AA/g
g of cells/g of bone1.68E-059.75E-05g of cells/g of mudstone
g dry wt/cell4.00E-144.00E-14g dry wt/cell
cells/gram4.19E + 082.44E + 09cells/gram
DNA
BoneMudstone
ng/g79316.4ng/g
DNA g/g of bone7.93E-071.64E-08DNA g/g of mudstone
DNA g/cell3.00E-153.00E-15DNA g/cell
cells/g of bone2.64E + 085.47E + 06cells/g of mudstone
Appendix 1—table 27
Pairwise F values from PERMANOVA of species-level sequence percentages.
https://doi.org/10.7554/eLife.46205.103
MudstoneBone surface scrapingsBone coreEDTA demineralized bone core
Mudstone-17.6247.0546.28
Bone surface scrapings17.62-162.8168.6
Bone core47.05162.8-33.41
EDTA demineralized bone core46.28168.633.41-

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Sequencing data has been placed in the NCBI Sequence Read Archive under the accession number SRR7947417.

The following data sets were generated
  1. 1

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