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Chickenosaurus – Story

Dinosaurs haven’t all disappeared? Really?

Last update: April 2022

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Context

A mass extinction took place around 66 million years ago, in the spring, caused by the impact of the asteroid ‘Chicxulub impactor’ in Yucatan (Mexico).

This event provoked the disappearance of about 75% of species, including marine molluscs called ammonites and most dinosaurs and marine reptiles.

But not all dinosaurs disappeared: some survived. What have their descendants become?

Here are some pieces of the puzzle, based on small pieces of protein sequences from the fossilized bones of Brachylophosaurus canadensis and Tyrannosaurus rex, two species of dinosaurs which are thought to have disappeared some 80 and 68 million years ago, respectively!

Podcast: C’est au printemps que les dinosaures ont été rayés de la Terre (RTS, mars 2022) (FR) – Source: The Mesozoic terminated in boreal spring (2022)

A collection of fossils

Scientists collect information on the dinosaurs and the species which have disappeared based on different fossils from around the world: this allows them to recover morphological data and even, in some cases, molecular data (DNA or protein) that they can compare to determine the relationship that exists between the different species.

What is a fossil?

Fossils are traces, more or less mineralized, of ancient living organisms: they can be bones, teeth, eggs, traces of their past activity (footprints, coprolite, …) or castings, conserved in sedimentary rock.

And certain fossils still contain DNA or proteins!

Some key dates

Here is a brief summary that recaps the history of life on earth, of dinosaurs…and of the oldest fossils:

4,500 million years ago

The age of the earth, and that of the solar system, is estimated to be 4,500 million years, with an uncertainty of a few tens of million years.

 

Source: Comment a-t-on déterminé l’âge de la Terre ? (2021) (FR)

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3,500 to 4,000 million years ago

3,500 to 4,000 million years ago

The hypothetical ancestor common to all species is called LUCA (Last Universal Common Ancestor). It is believed to have lived 3,500 to 4,000 million years ago and have been composed of only one cell.

 

Source: The physiology and habitat of the last universal common ancestor (2016) / Physiology, phylogeny, and LUCA (2016)

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3,500 to 3,800 million years ago

Among the most ancient fossils, one finds those of unicellular organisms which are thought to have lived some 3,500 milion years ago. Traces of their presence were found in 1993 in Australia. Other traces of life dating from 3,800 million years ago were found in Quebec in 2017.

Source: Evidence for early life in Earth’s oldest hydrothermal vent precipitates (2017) / SIMS analyses of the oldest known assemblage of microfossils document their taxon-correlated carbon isotope compositions (2017) / Metabolically diverse primordial microbial communities in Earth’s oldest seafloor-hydrothermal jasper (2022) 

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635 million years ago

Fossile terrestre

The oldest land fossil was recovered in China in 2021. It is a kind of fungus which is thought to have lived some 635 million years ago. (Image: Andrew Czaja of University of Cincinnati)

Source: Cryptic terrestrial fungus-like fossils of the early Ediacaran Period (2021)

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425 million years ago

The fossil of a centipede, Kampecaris obanensis, which is thought to have lived some 425 million years ago, was discovered on a Scottish island.

Source: Myriapod divergence times differ between molecular clock and fossil evidence (2020)

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150 million years ago

150 million years ago

In 1861, a fossilized feather slightly less than 7 cm in length and about 150 million years old, was discovered in the quarries in Solnhofen, Germany. Who was the owner?

Source: Detection of lost calamus challenges identity of isolated Archaeopteryx feather (2019)

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80 million years ago

Brachylophosaurus

The oldest fragments of proteins found to date are derived from the collagen of Brachylophosaurus canadensis. This species of dinosaurs which is thought to have lived 80 million years ago, was discovered in Montana (USA) in 2009.

 

>Brachylophosaurus canadensis – collagen COL1A1 
GATGAPGIAGAPGFPGARGPSGPQGPSGAPGPK GVQGPPGPQGPRGLT GPIGPTGFPGAA

 

Source: Expansion for the Brachylophosaurus canadensis Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein (2017) / 80-Million-Year-Old Dinosaur Collagen Confirmed (2017)

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68 million years ago

T-Rex

Two teams were able to identify collagen fragments from the femur of a 68-million-year-old Tyrannosaurus rex. This specimen was discovered in California. The collagen fragments were analysed for the first time in 2007.

Source: Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry (2007) / Reanalysis of Tyrannosaurus rex Mass Spectra (2009)

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66 million years ago

The fifth mass extinction which led to the disappearance of about 75% of species, including marine molluscs called ammonites and most dinosaurs and marine reptiles.

Source : Wikipedia

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1.25 to 2.08 million years ago

mammoth

The oldest DNA sequenced to date was collected on three molars belonging to 3 mammoths which lived some 1.25 to 2.08 million years ago. These molars were recovered in north eastern Siberia.

>DQ1888299.2 Mammuthus primigenius mitochondrion, complete genome
GTTAATGTAGCTTAAAACAAAAGCAAGGTACTGAAAATACCTAGACGAGTATATCCAACTCCATAAACAACAAAGGTTTGGTCCCGGCCTTCTTATTGGTTACTAGGAAACTTATACATGCAAGTATCCGCCCGCCAGTGAATACGCCTTCTAAATCATCACTGATCAAAGAGAGCTGGCATCAAGCACACACCCCAAGTGTAGCTCATGACGTCTCGCCTAGCCACACCCCCACGGGAAACAGCAGTAGTAAATATTTAGCAATTAACAAAAGTTAGAC...

 

Source: Million-year-old DNA sheds light on the genomic history of mammoths (2021)

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Fossilized proteins?

It is nowadays possible to sequence the proteins found in the fossils of eggshells, teeth, bones, or mummified tissues: this field is referred to as ‘Deep Time Paleoproteomics’ …and the records are starting to accumulate as proteins appear to be better preserved than DNA over time and technologies are improving more and more.

Source: Deep Time Paleoproteomics: Looking Forward (2022)

Sequencing a protein?

Like a pearl necklace, a protein consists of a chain of amino acids bound to each other by chemical bonds. There are 20 different amino acids, represented by the letters (G, E, N, I , A, L, …).

Mass spectrometry is a technology which allows proteins to be sequenced, that is, to determine the order of the amino acids in the chain.

Famous proteins in palaeontology…

Collagen and cytochrome are proteins studied by scientists interested in organisms which have disappeared.

Why?

Collagen is one of the most abundant proteins in bones. This protein is the one most frequently found in the organic material present in fossilized bones.

Here is the collagen protein from different species and the collagen protein of species which today have disappeared from the UniProtKB database.

The information that we have on certain proteins also comes from DNA. If the DNA sequence is known, the corresponding protein sequence can be predicted. DNA is found primarily in the nucleus of eukaryotic cells, but also in organelles such as mitochondria or chloroplasts. However, the DNA found in the mitochondria is better preserved over time than the DNA present in the nuclei of cells. The cytochrome protein is one of the proteins whose gene is found in mitochondrial DNA.

For example, here is the mitochondrial DNA of Neanderthal man and the sequence of the cytochrome protein of Neanderthal man or, for instance, the sequence of the cytochrome proteins of the dodo bird and of the Tasmanian wolf.

Here is the list of proteins from species which are extinct today from the UniProtKB database.

Here are examples of collagen fragments found for different species.

Tyrannosaurus rex

T-rex

GATGAPGIAGAPGFPGARGAPGPQGPSGAPGPKGSAGPPGATGFPGAAGRGVQGPPGPQGPR

Dating: 68 million years

Gallus gallus (chicken)

gallus gallus (poulet)

GATGAPGIAGAPGFPGARGPSGPQGPSGAPGPKGNSGEPGAPGNKGDTGAKGEPGPAGVQGP

    Dating: 10,000 to 50,000 years

 

Mammut americanum (mammoth)

Mammouth

GANGAPGIAGAPGFPGARGPAGPQGPSGAPGPKGNSGEPGAPGSKGDAGAGIQGPPGPAGEE

Dating: 1.65 million years

 

Brachylophosaurus canadensis

Brachylophosaurus canadensis

GATGAPGIAGAPGFPGARGPSGPQGPSGAPGPKGSAGPPGATGFPGAAGRGETGPAGPAGPP

Dating: 80 million years

 

The evolution of collagen

To define parentage, biologists search not only for what species have in common, but also for what distinguishes them from one another.

The collagen protein is present, among other places, in the bones of all vertebrates. But as is the case for all proteins, it is a bit different from one species to another. These differences are a real gold mine to study the evolution and the relationship that exists between the different species.

How can one study the relationship between different species?

One can, for instance, count the differences between the collagen sequences from the different species and thus estimate which species are the closest.

The collagen sequences from Brachylophosaurus canadensis and Gallus gallus (chicken) have been aligned below: the presence of an * means that the amino acids are identical.

How many differences are there?

 

This approach is a bit tedious but, more importantly, it is not quite complete: certain sequence changes have a greater biological impact than others. It’s a bit like a text: some spelling errors will modify the sense of the text while others do not.

This is the reason why bioinformatic programs which compare sequences use ‘substitution matrices’.

A value is attributed to each pair of amino acids (for example: A-A: 4; G-G: 0; A-R: – 1). The sum of these values enables the score of the alignment to be calculated.

For the experts: an example of a substitution matrix

Substitution matrices allow the score of an alignment to be calculated by giving a value to each ‘pair’ of amino acids. Different substitution matrices exist which have been established by studying the evolution of different protein families.

In general, the substitution value is based on the degree of similarity of the two amino acids. If they are very similar (in terms of shape and physicochemical properties), the amino acid pair is probable and will therefore receive a positive score. If the amino acids are very different, the pair is unlikely and will therefore receive a negative score. A score of 0 corresponds to a pair that could be observed by chance.

For example, here is the substitution matrix called BLOSUM62:

Classification of dinosaurs and birds: an example

The tree below has been calculated by comparing the sequences of different collagen proteins from over 30 different species. It has been validated by complex statistical programs.

Image freely adapted from Expansion for the Brachylophosaurus canadensis Collagen I Sequence and Additional Evidence of the Preservation of Cretaceous Protein (2017)

These analyses have made it possible to establish that dinosaurs are closely related to birds.

These results confirm predictions made based on the anatomical comparison of skeletons. However, this is the first molecular (genetic) proof of the evolutionary link that exists between modern birds and dinosaurs.

Dinosaurs have thus not all disappeared: a small group survived and among these, the ‘avian’ dinosaurs, the ancestors of birds.

Another more detailed tree to understand the relationship between birds and dinosaurs (wikimedia).

The mystery of the feather

In 1861, a few months after the publication of Charles Darwin’s work On the Origin of Species, a fossilized feather slightly less than 7 cm in length and about 150 million years old, was discovered in the quarries in Solnhofen, Germany. The identification of the owner of the feather has long been the object of controversy. According to the latest studies, it is thought to have belonged to Archaeopteryx lithographica, a feathered dinosaur.

Source: Detection of lost calamus challenges identity of isolated Archaeopteryx feather (2019)

 

Today we know that Archaeopteryx lithographica is more related to birds than to other dinosaurs. The classification of dinosaurs and birds, however, still remains controversial!

Some 150 million years ago, Archaeopteryx lithographica belonged to a species among many, a species with no programmed destiny!

Archaeopteryx lithographica Source: Wikipedia

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