Ancient Rhino tooth helps push the boundaries of evolutionary research
University of York
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Ancient rhino tooth
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Scientists have shed new light on the rhino family tree after recovering a protein sequence from a fossilised tooth from more than 20 million years ago.
The recovered protein sequences allowed researchers to determine that this ancient rhino diverged from other rhinocerotids during the Middle Eocene-Oligocene epoch, around 41-25 million years ago.
The data also shed new light on the divergence between the two main subfamilies of rhinos, Elasmotheriinae and Rhinocerotinae, suggesting a more recent split in the Oligocene, around 34-22 million years ago, than shown previously through bone analysis.
The successful extraction and sequencing of ancient enamel proteins from a fossilized rhino tooth extends the timescale for recoverable, evolutionary-informative protein sequences by ten-fold compared to the oldest known ancient DNA.
The team at York were involved in confirming that the proteins and amino acids were genuinely ancient. They analysed the rhino tooth, which was unearthed in Canada's High Arctic, using a technique known as chiral amino acid analysis to gain a clearer understanding of how the proteins within it had been preserved.
By measuring the extent of protein degradation and comparing it to previously analysed rhino material, they were able to confirm that the amino acids were original to the tooth and not the result of later contamination.
Dr Marc Dickinson, co-author and postdoctoral researcher at the University of York’s Department of Chemistry, said: “It is phenomenal that these tools are enabling us to explore further and further back in time. Building on our knowledge of ancient proteins, we can now start asking fascinating new questions about the evolution of ancient life on our planet.”
The rhino is of particular interest as it is now classified as an endangered species, and so understanding its deep-time evolutionary history, allows us to gain vital insights into how past environmental changes and extinctions shaped the diversity we see today.
To date, scientists have relied on the shape and structure of fossils or, more recently, ancient DNA (aDNA) to piece together the evolutionary history of long-extinct species. However, aDNA rarely survives beyond 1 million years, limiting its utility for understanding deep evolutionary past.
While ancient proteins have been found in fossils from the Middle-Late Miocene, - roughly the last 10 million years - obtaining sequences detailed enough for robust reconstructions of evolutionary relationships was previously limited to samples no older than four million years.
The new study, published in the journal Nature, significantly expands that window, demonstrating the potential of proteins to persist over vast geological timescales under the right conditions.
Fazeelah Munir, who analysed the tooth as part of her doctoral research at the University of York’s Department of Chemistry, said: “Successful analysis of ancient proteins from such an old sample gives a fresh perspective to scientists around the globe who already have incredible fossils in their collections. This important fossil helps us to understand our ancient past.”
The fossil was in a region of Canada currently characterized by permafrost, and researchers say that dental enamel and the relatively cold environment the fossil was found in, played an important part in the long preservation of the proteins.
Dental enamel provides a stable ‘scaffold’ that can protect ancient proteins from degradation over geological time. The hardness of enamel, which results from a complex structure of minerals, acts as a protective barrier, slowing down the breakdown of proteins that occurs after death.
Professor Enrico Cappellini, from Globe Institute, University of Copenhagen, said: "The Haughton Crater may be a truly special place for palaeontology: a biomolecular vault protecting proteins from decay over vast geological timescales.
“Its unique environmental history has created a site with exceptional preservation of ancient biomolecules, akin to how certain sites preserve soft tissues. This finding should encourage more paleontological fieldwork in regions around the world."
Ryan Sinclair Paterson, postdoctoral researcher at the Globe Institute, University of Copenhagen, added: “This discovery is a game-changer for how we can study ancient life.”
Journal
Nature
Long in the tooth
18 million years of protein enamel uncovered
By Clea Simon/Harvard Correspondent
Proteins degrade over time, making their history hard to study. But new research has uncovered ancient proteins in the enamel of the teeth of 18-million-year-old fossilized mammals from Kenya’s Rift Valley, opening a window into how these animals lived and evolved.
In their new paper in Nature, researchers from Harvard and the Smithsonian Museum Conservation Institute discuss their findings. “Teeth are rocks in our mouths,” explained Daniel Green, field program director in the Department of Human Evolutionary Biology and the paper’s lead author. “They're the hardest structures that any animals make, so you can find a tooth that is a hundred or a hundred million years old, and it will contain a geochemical record of the life of the animal.” That includes what the animal ate and drank, as well as its environment.
“In the past we thought that mature enamel, the hardest part of teeth, should really have very few proteins in it at all,” said Green. However, utilizing a new newer proteomics technique called liquid chromatography tandem mass spectrometry (LC-MS/MS), the team was able to detect a “a great diversity of proteins. . . . in different biological tissues.”
“The technique involves several stages where peptides are separated based on their size or chemistry so that they can be sequentially analyzed at higher resolutions than was possible with previous methods,” explained Kevin T. Uno, associate professor in HEB and one of the paper’s corresponding authors.
“We and other scholars recently found that there are dozens – if not even hundreds – of different kinds of proteins present inside tooth enamel,” said Green.
With the realization that many proteins are found in contemporary teeth, the researchers turned to fossils, collaborating with the Smithsonian and the National Museum of Kenya for access to fossilized teeth, particularly those of early elephants and rhinos. As herbivores, they had large teeth for grinding their diet of plants. These mammals, continued Green, “can have enamel two to three millimeters thick. It was a lot of material to work with.”
What they found – peptide fragments, chains of amino acids, that together form proteins as old as 18 million years – was “field-changing,” according to Green. “Nobody’s ever found peptide fragments that are this old before,” he said, calling the findings “kind of shocking.” Until now, the oldest published materials are about three and a half million years old, he said. “With the help of our colleague Tim Cleland, a superb paleoproteomicist at the Smithsonian, we’re pushing back the age of peptide fragments by five or six times what was known before.”
The newly discovered peptides cover a range of proteins that perform different functions, altogether known as the proteome, Green said.
“One of the reasons that we’re excited about these ancient teeth is that we don't have the full proteome of all proteins that could have been found inside the bodies of these ancient elephants or rhinoceros, but we do have a group of them.” With such a collection, “there might be more information available from a group of them than just one protein by itself.”
This research “opens new frontiers in paleobiology, allowing scientists to go beyond bones and morphology to reconstruct the molecular and physiological traits of extinct animals and hominins,” said Emmanuel K. Ndiema, senior research scientist at the National Museum of Kenya, and paper coauthor. “This provides direct evidence of evolutionary relationships. Combined with other characteristics of teeth, we can infer dietary adaptations, disease profiles, and even age at death – insights that were previously inaccessible.”
In addition to shedding light on the lives of these creatures, it helps place them in history. Uno elaborated: “We can use these peptide fragments to explore the relationships between ancient animals, similar to how modern DNA in humans is used to identify how people are related to one another.”
“Even if an animal is completely extinct – and we have some animals that we analyze in our study who have no living descendants – you can still, in theory, extract proteins from their teeth and try to place them on a phylogenetic tree,” said Green. Such information “might be able to resolve longstanding debates between paleontologists about what other mammalian lineages these animals are related to using molecular evidence.”
Although this research began as “a small side project” of a much larger project involving dozens of institutions and researchers from around the world, said Green, “we were surprised at just how much we found. There really are a lot of proteins preserved in these teeth.”
This research was partially funded by the National Science Foundation and Smithsonian’s Museum Conservation Institute.
IMAGES AND EMBARGOED DRAFT OF PAPER AVAILABLE AT THIS LINK:
Journal
Nature
Article Title
Eighteen million years of diverse enamel proteomes from the East African Rift
Article Publication Date
9-Jul-2025
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