Protein is the clue to solving a Darwinian mystery

Bone collagen sequences prove that South American native ungulates are closely related to horses, rhinos and tapirs but not to elephants

March 18, 2015

The South American native ungulates Toxodon and Macrauchenia were first found by Darwin 180 years ago in Uruguay and Argentina, yet their place in the phylogenetic tree of the mammals has long been an issue of debate. An international team of scientists from the University of York and the Natural History Museum in London, UK, the American Museum of Natural History in New York, USA, and the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, has now provided the key to solving the evolutionary puzzle surrounding what Charles Darwin called the ‘strangest animals ever discovered’. Their remarkable technical feat in obtaining a molecular phylogeny based on Pleistocene protein sequences is a first, which could herald a new chapter in palaeontology.

A team of researchers was called in by the Natural History Museum and the American Museum of Natural History to resolve the long-standing question of the fossils of Toxodon and Macrauchenia, the South American native ungulates (SANUs) first found by Darwin 180 years ago in Uruguay and Argentina. It was unclear if the SANUs had a single origin or several, whether or not they arose before or after the Cretaceous period and whether they belonged to the order of mammals which includes elephants or that which includes cattle and horses.

Previously, attempts by scientists to pinpoint the origin of SANUs using morphology-based analysis and ancient DNA, had failed. The latter approach was compromised because scientists were unable to recover any identifiable mammalian DNA from fossil specimens. This is likely to be the case for large numbers of important fossils from tropical or temperate deposits, as DNA preservation is ultimately controlled by the thermal history of material.

But the structural protein, collagen, is likely to survive around ten times longer than DNA so the scientists used proteomic analysis to screen 48 fossil bone samples of Toxodon and Macrauchenia discovered in the 19th century in the same area as those recovered by Darwin.  This produced sequences covering more than 90 per cent of the collagen molecule, effectively providing a phylogenetic barcode for the two species. The collagen sequences proved that Toxodon and Macrauchenia are closely related to horses, rhinos and tapirs. “By producing the most comprehensive example of sequencing of its type, we have been able to resolve the taxonomic placement of these mammals, and solve a question that has baffled palaeontologists for more than a century”, says Jessica Thomas of York University.

“We now have the potential to address many more of these challenges and to explore the evolutionary process much further back in prehistory”, says Matthew Collins, who headed the York research team. And PhD student Frido Welker of the Max Planck Institute for Evolutionary Anthropology and the University of York adds: “We are developing new proteomic techniques and applying them to the archaeological and palaeoanthropological record. To get 90 per cent sequence coverage for both species is a direct result of those efforts, and truly fantastic given the burial time and location.”

“Our long-standing collaboration with scientists at Bruker Daltonics made it possible to analyse these samples using the most up to date mass spectrometric instrumentation. The outstanding quality of the data was crucial in enabling us to determine the collagen sequences”, says Jane Thomas-Oates, director of York’s Centre of Excellence in Mass Spectrometry.

“In addition to ancient DNA sequencing, the analysis of fossil proteins offers an entirely new approach to decipher the evolution of extinct mammals, including hominins”, says Jean-Jacques Hublin, director at the Max Planck Institute for Evolutionary Anthropology.


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