Mary H. Schweitzer

Biomolecular characterization and protein sequences of the Campanian hadrosaur B. canadensis.

The Birds and the Dinosaurs The extent to which primary tissues are preserved in ancient fossils remains controversial. Schweitzer et al. (p. 626; see the news story by Service) describe well-preserved tissues and primary collagen sequences from the femur of an 80-million-year-old hadrosaur. The fossil preserved structures resembling primary bone tissues and vessels. Both extracts and tissue pieces were analyzed in multiple laboratories by mass spectrometry, which revealed ancient collagen sequences that support a close relation between birds and dinosaurs. Analysis of well-preserved tissues from an 80-million-year-old hadrosaur supports the dinosaur-bird relationship. Molecular preservation in non-avian dinosaurs is controversial. We present multiple lines of evidence that endogenous proteinaceous material is preserved in bone fragments and soft tissues from an 80-million-year-old Campanian hadrosaur, Brachylophosaurus canadensis [Museum of the Rockies (MOR) 2598]. Microstructural and immunological data are consistent with preservation of multiple bone matrix and vessel proteins, and phylogenetic analyses of Brachylophosaurus collagen sequenced by mass spectrometry robustly support the bird-dinosaur clade, consistent with an endogenous source for these collagen peptides. These data complement earlier results from Tyrannosaurus rex (MOR 1125) and confirm that molecular preservation in Cretaceous dinosaurs is not a unique event.

Remarkable Preservation of Undigested Muscle Tissue Within a Late Cretaceous Tyrannosaurid Coprolite from Alberta, Canada

Abstract Exceptionally detailed soft tissues have been identified within the fossilized feces of a large Cretaceous tyrannosaurid. Microscopic cord-like structures in the coprolitic ground mass are visible in thin section and with scanning electron microscopy. The morphology, organization, and context of these structures indicate that they are the fossilized remains of undigested muscle tissue. This unusual discovery indicates specific digestive and taphonomic conditions, including a relatively short gut-residence time, rapid lithification, and minimal diagenetic recrystallization. Rapid burial of the feces probably was facilitated by a flood event on the ancient coastal lowland plain on which the fecal mass was deposited.

Soft tissue and cellular preservation in vertebrate skeletal elements from the Cretaceous to the present

Soft tissues and cell-like microstructures derived from skeletal elements of a well-preserved Tyrannosaurus rex (MOR 1125) were represented by four components in fragments of demineralized cortical and/or medullary bone: flexible and fibrous bone matrix; transparent, hollow and pliable blood vessels; intravascular material, including in some cases, structures morphologically reminiscent of vertebrate red blood cells; and osteocytes with intracellular contents and flexible filipodia. The present study attempts to trace the occurrence of these four components in bone from specimens spanning multiple geological time periods and varied depositional environments. At least three of the four components persist in some skeletal elements of specimens dating to the Campanian. Fibrous bone matrix is more altered over time in morphology and less likely to persist than vessels and/or osteocytes. Vessels vary greatly in preservation, even within the same specimen, with some regions retaining pliability and other regions almost crystalline. Osteocytes also vary, with some retaining long filipodia and transparency, while others present with short and stubby filipodia and deeply pigmented nuclei, or are pigmented throughout with no nucleus visible. Alternative hypotheses are considered to explain the origin/source of observed materials. Finally, a two-part mechanism, involving first cross-linking of molecular components and subsequent mineralization, is proposed to explain the surprising presence of still-soft elements in fossil bone. These results suggest that present models of fossilization processes may be incomplete and that soft tissue elements may be more commonly preserved, even in older specimens, than previously thought. Additionally, in many cases, osteocytes with defined nuclei are preserved, and may represent an important source for informative molecular data.

Late Cretaceous avian eggs with embryos from Argentina

MARY H. SCHWEITZER1,2, FRANKIE D. JACKSON2, LUIS M. CHIAPPE3, JAMES G. SCHMITT2, JORGE O. CALVO4, and DAVID E. RUBILAR5 1Department of Microbiology, Montana State University, Bozeman, Montana 59717, schweitz@montana.edu; 2Department of Earth Sciences and Museum of the Rockies, Montana State University, Bozeman, Montana 59717; 3Natural History Museum of Los Angeles, Los Angeles, California 90007; 4Museo de Geologia y Paleontologia, Universidad Nacional del Comahue, Neuqu6n, Argentina; 5Instituto de Geociencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile, and Secci6n Paleontologia, Museo Nacional de Historia Natural, Casilla 787, Santiago, Chile

Molecular Phylogenetics of Mastodon and Tyrannosaurus rex

We report a molecular phylogeny for a nonavian dinosaur, extending our knowledge of trait evolution within nonavian dinosaurs into the macromolecular level of biological organization. Fragments of collagen α1(I) and α2(I) proteins extracted from fossil bones of Tyrannosaurus rex and Mammut americanum (mastodon) were analyzed with a variety of phylogenetic methods. Despite missing sequence data, the mastodon groups with elephant and the T. rex groups with birds, consistent with predictions based on genetic and morphological data for mastodon and on morphological data for T. rex. Our findings suggest that molecular data from long-extinct organisms may have the potential for resolving relationships at critical areas in the vertebrate evolutionary tree that have, so far, been phylogenetically intractable.

Melanosomes or Microbes: Testing an Alternative Hypothesis for the Origin of Microbodies in Fossil Feathers

Microbodies associated with fossil feathers, originally attributed to microbial biofilm, have been reinterpreted as melanosomes: pigment-containing, eukaryotic organelles. This interpretation generated hypotheses regarding coloration in non-avian and avian dinosaurs. Because melanosomes and microbes overlap in size, distribution and morphology, we re-evaluate both hypotheses. We compare melanosomes within feathers of extant chickens with patterns induced by microbial overgrowth on the same feathers, using scanning (SEM), field emission (FESEM) and transmission (TEM) electron microscopy. Melanosomes are always internal, embedded in a morphologically distinct keratinous matrix. Conversely, microbes grow across the surface of feathers in continuous layers, more consistent with published images from fossil feathers. We compare our results to both published literature and new data from a fossil feather ascribed to Gansus yumenensis (ANSP 23403). ‘Mouldic impressions’ were observed in association with both the feather and sediment grains, supporting a microbial origin. We propose criteria for distinguishing between these two microbodies.

Identification of Immunoreactive Material in Mammoth Fossils

The fossil record represents a history of life on this planet. Attempts to obtain molecular information from this record by analysis of nucleic acids found within fossils of extreme age have been unsuccessful or called into question. However, previous studies have demonstrated the long-term persistence of peptides within fossils and have used antibodies to extant proteins to demonstrate antigenic material. In this study we address two questions: Do immunogenic/antigenic materials persist in fossils? and; Can fossil material be used to raise antibodies that will cross-react with extant proteins? We have used material extracted from a well-preserved 100,000–300,000-year-old mammoth skull to produce antisera. The specificity of the antisera was tested by ELISA, western blotting, and immunohistochemistry. It was demonstrated that antisera reacted specifically with the fossils and not the surrounding sediments. Reactivity of antisera with modern proteins and tissues was also demonstrated, as was the ability to detect evolutionary relationships via antibody–antigen interactions. Mass spectrometry demonstrated the presence of amino acids and specific peptides within the fossil. Peptides were purified by anion-exchange chromatography and sequenced by tandem mass spectrometry. The collagen-derived peptides may have been the source of at least some of the immunologic reactivity, but the antisera identified molecules that were not observed by mass spectrometry, indicating that immunologic methods may have greater sensitivity. Although the presence of peptides and amino acids was demonstrated, the exact nature of the antigenic material was not fully clarified. This report demonstrates that antibodies may be used to obtain information from the fossil record.

Preservation of biomolecules in cancellous bone of Tyrannosaurus rex

ABSTRACT An exceptionally well preserved specimen of the tyrannosaurid dinosaur Tyrannosaurus rex Osborn shows little evidence of permineralization or other diagenetic effects. It appears that the cancellous bone tissues of the specimen may have been protected from water infiltration or mineral deposition by the very dense cortical bone which surrounds them. The cancellous tissues provided an opportunity to test the hypothesis that indigenous biomolecules might be preserved over the course of millions of years under the appropriate conditions. HPLC analysis of extracts from the bone tissues revealed the presence of molecules with light absorbance maxima consistent with nucleic acids and peptides/proteins. Analyses of bone extracts for amino acid content yielded ratios similar to those found for modern ostrich and horse bone. A high molar glycine ratio and the presence of hydroxylysine peaks in bony tissue samples from the T. rex suggests the presence of collagen type I remnants. Results indicate that the ...

A role for iron and oxygen chemistry in preserving soft tissues, cells and molecules from deep time

The persistence of original soft tissues in Mesozoic fossil bone is not explained by current chemical degradation models. We identified iron particles (goethite-αFeO(OH)) associated with soft tissues recovered from two Mesozoic dinosaurs, using transmission electron microscopy, electron energy loss spectroscopy, micro-X-ray diffraction and Fe micro-X-ray absorption near-edge structure. Iron chelators increased fossil tissue immunoreactivity to multiple antibodies dramatically, suggesting a role for iron in both preserving and masking proteins in fossil tissues. Haemoglobin (HB) increased tissue stability more than 200-fold, from approximately 3 days to more than two years at room temperature (25°C) in an ostrich blood vessel model developed to test post-mortem ‘tissue fixation’ by cross-linking or peroxidation. HB-induced solution hypoxia coupled with iron chelation enhances preservation as follows: HB + O2 > HB − O2 > −O2 ≫ +O2. The well-known O2/haeme interactions in the chemistry of life, such as respiration and bioenergetics, are complemented by O2/haeme interactions in the preservation of fossil soft tissues.

Keratin immunoreactivity in the Late Cretaceous bird Rahonavis ostromi

ABSTRACT Immunohistochemical studies, supported by additional lines of evidence, suggest that original proteinaceous components of keratin claw sheath material may be preserved in the pedal ungual phalanx associated with the primitive bird, Rahonavis ostromi, from the Late Cretaceous of Madagascar. This conclusion is based upon immunohistochemical analyses, and supported by brightfield, transmission, and scanning electron microscopy, mass spectrometry, and x-ray photoelectron spectroscopy. Although keratinous structures such as hair, nails, claws, scales and feathers have been identified in the fossil record, these identifications were based on morphological similarity rather than molecular analyses. Chemical or immunological evidence for the survival of keratin has not previously been established in fossils older than ~33,000 BP. This study demonstrates immunological staining and amino acid composition consistent with the presence of fragments of beta keratin, a protein family unique to reptiles and bird...