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New method reveals the secrets of evolution

Researchers could only previously map genetic relationships in the past 700,000 years. Danish researchers have now pushed back this time scale to 1.7 million years.

Researchers have become more skilled at revealing the secrets of evolution in recent years.

Sequencing ancient DNA has enabled researchers to determine the origin of dogs or how humans and Neanderthals are related.

However, researchers are limited in how far they can look back in time by how long DNA can survive in fossils. DNA does not last forever, and so far the most ancient DNA has been found in the permafrost areas in Canada and is 700,000 years old.

Most DNA, however, is not that old because preserving it until the present requires permafrost over many thousands of years.

However, this problem may be solved after researchers from the University of Copenhagen found that they can instead use ancient proteins to determine how various long-extinct species of animals are related to their modern relatives. Researchers have used the new method and so far managed to reach an astonishing 1.7 million years back in time. However, there is so much more potential.

“Proteins survive much better in fossils than does DNA, so this is huge progress in looking even further back in time than we have ever been able to before,” explains a researcher behind the development of the new method, Enrico Cappellini, Associate Professor, Section for Evolutionary Genomics, Globe Institute, University of Copenhagen.

The method was recently presented in Nature.

Proteins in dental enamel are extremely well preserved

This method is not that different from other methods for identifying amino acid sequences in proteins.

Researchers routinely use mass spectrometry to determine how individual amino acids in a protein are positioned in relation to each other, and by comparing the amino acid sequence of a given protein from, for example, a living person, a chimpanzee, a gorilla and an orangutan, researchers can determine how the species are related and which species first diverged from the others evolutionarily.

The revolutionary aspect of the new method is that the researchers investigate proteins in dental enamel.

Dental enamel is the hardest material in animals, and enamel is very good at preserving proteins over time.

“Analysing proteins does not provide the same genetic information as DNA, but we can look much further back in time to eras from which no DNA remains. So far we have gone 1.7 million years back in time, but we do not know how far this method can take us back,” explains Enrico Cappellini.

Ancient protein should not be treated with enzymes

Enrico Cappellini and colleagues have developed the method of extracting and analysing ancient proteins.

The key aspect of this method relative to the techniques currently used for analysing proteins is that researchers do not initially degrade the protein.

When researchers analyse a protein, they typically add an enzyme that degrades the protein so that the separate elements can be analysed individually and subsequently compiled as an amino acid puzzle of the overall amino acid sequence.

However, Enrico Cappellini and colleagues found that they did not need to add the enzymes before analysing the samples with mass spectrometry because the proteins in ancient dental enamel have already been fragmented by the passage of time.

“The difficult part of the process was not the analysis itself but determining which material to use to find the proteins and how to isolate the proteins to analyse them. Every aspect of the normal way of analysing proteins had to be adjusted and adapted for these ancient proteins,” says Enrico Cappellini.

Mapped relationships for an ancient rhinoceros

Enrico Cappellini says that the new technique has extremely interesting perspectives.

Researchers have used the technique to map the sequence of amino acids in proteins from an ancient rhinoceros that lived 1.7 million years ago in Dmanasi, located in present-day Georgia.

The amino acid sequence was 900 amino acids long and almost complete.

The researchers compared this amino acid sequence with similar amino acid sequences from the dental enamel of five modern rhinoceroses (Sumatra, Java, Indian, white and black) and two extinct rhinoceros species.

The researchers used this comparison to establish that the rhinoceros from Dmanasi was a sister group to the two extinct rhinoceroses and also determined how it was related to modern rhinoceroses.

“Both the Dmanasi rhinoceros and the modern rhinoceroses had a common ancestor that lived in Europe and western Asia more than 2 million years ago. The Dmanasi rhinoceros diverged from the evolutionary tree first, with the other two extinct rhinoceroses diverging later,” explains Enrico Cappellini.

The reconstruction of the proteins also indicates that, among modern rhinoceroses, the Sumatra rhinoceros is most closely related to the Dmanasi rhinoceros, although not a direct relative.

Investigating human ancestors

How long-extinct rhinoceroses are related to each other may not be of great interest to the general public, and rhinoceroses are not Enrico Cappellini’s future focus.

The perspectives are much greater.

For example, the researchers are already examining the potential of using this technique to determine how our ancestors are related.

Human evolutionary history has included many human species, some of which became extinct in evolutionary dead-ends, whereas others led to modern humans.

The entire puzzle of mapping the relationships of these long-extinct human relatives has so far been based solely on morphological studies of occasional findings of skulls and bones excavated in Africa and Asia.

Studies of proteins in dental enamel found by palaeontologists may shed new light on our history and reveal who our direct ancestors were and who our distant aunts and uncles were.

“This is naturally the most interesting perspective in applying this new technique to investigate and compare ancient proteins,” says Enrico Cappellini.

Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny” has been published in Nature. Several co-authors are employed at the Novo Nordisk Foundation Center for Protein Research, University of Copenhagen.

Enrico Cappellini
Lektor v. EvoGenomics
I use high-resolution mass spectrometry (MS) to sequence ancient protein residues recovered from paleontological and cultural heritage materials. I am actively involved in methodological development to push reliable recovery of ancient proteins further back in time, to minimise starting sample quantities and to improve data analysis and interpretation. Initially trained in biochemistry and molecular biology at the University of Turin (Italy), I then expanded my expertise in proteomics and investigation of ancient proteins and DNA. I was trained in analysis of ancient DNA from archaeological human remains (Cappellini et al., Journal of Archaeological Science 2004) and I received my PhD at the University of Florence (Italy) in 2003. During my post-doctoral activity in at the University of York (UK) I continued working on ancient DNA and started characterisation of ancient protein residues by amino acid racemisation analysis and MS-based proteomics (Willerslev, Cappellini et al., Science 2007, Cappellini et al., Naturwissenschaften 2010). I moved to the Centre for GeoGenetics, Natural History Museum of Denmark (SNM), University of Copenhagen (DK), in 2009 and I continued applying proteomics techniques to ancient samples in collaboration with colleagues at the Novo Nordisk Foundation center for Protein Research (CPR). We published published the first ancient proteome (Cappellini et al., JPR, 2012) and the first ancient oral metaproteome (Warinner et al., Nature Genetics, 2014). As a demonstration of his prominent role in paleoproteomics investigation he was invited to submit a “Perspective” piece by the multidisciplinary scientific magazine “Science”. In October 2016 I was appointed Associate Professor in Paleoproteomics at SNM to start my own research group.