Cretaceous collagen: Can molecular paleontology glean soft tissue from dinosaurs?
Mary Schweitzer, of North Carolina State University, and her colleagues first reported the discovery of soft tissue in a
Tyrannosaurus rex femur in 2005. The vial contains a sample of the bone. Credit: both: ©Science.
In the early 1990s, “Jurassic Park” hatched a wild idea: that
dinosaurs could be reincarnated using tiny amounts of preserved DNA. In
the bestselling books and blockbuster movies, the premise hinged on
extracting fragments of dinosaur DNA from ancient blood-sucking insects
preserved in amber, filling in the genetic gaps with snippets of modern
amphibian DNA and hatching the engineered dinosaurs out of ostrich eggs.
In reality, however, while many amazing things have been found in
fossilized tree sap, DNA isn’t one of them.
“Jurassic Park” fans saw a glimmer of hope in 2005, when a team of molecular paleontologists led by
Mary Schweitzer of North Carolina State University
reported finding soft tissue preserved inside a 68-million-year-old
Tyrannosaurus rex
femur. DNA is notoriously delicate, and dinosaur DNA itself has not
been found — a possibility Schweitzer calls extremely unlikely since the
oldest DNA recovered to date is less than a million years old and
nonavian dinosaurs died out 66 million years ago. However, in the decade
since that initial find, Schweitzer’s team has unearthed mounting
evidence that soft tissues, such as blood vessels, collagen and other
proteins, whose long, folded chains of amino acids make them much more
robust than DNA, can survive more than 66 million years of degradation.
If 68-million-year-old dinosaur soft tissue can really be recovered —
a claim that has been met with much skepticism — it may help
paleontologists answer some long-standing questions about dinosaur
physiology, such as whether the behemoths were cold- or warm-blooded.
The existence of ancient preserved soft tissues would also change how we
think about fossilization and the handling and storing of fossils:
After all, if soft tissue can be recovered from ancient fossils, we need
to be more careful in how we extract and preserve them. Living, roaring
tyrannosaurs may not be the future, but perhaps molecular paleontology
is.
An Accidental Find
Schweitzer working in a lab at Montana State University. Her team has
found evidence for soft tissues in about one-third of the several dozen
dinosaur specimens they have studied. Credit: Kelly Gorham, Montana
State University.
The young field of molecular paleontology was thrust into the
international spotlight in 2005 when Schweitzer’s team announced in
Science their discovery of
T. rex soft tissue. The tissues were
found by accident: After some broken femur fragments were dissolved in a
weak acid solution, a scrap of oddly stretchy material was left over.
Under a microscope, the scrap showed what appeared to be bifurcating
blood vessels, cellular structures similar to those found in bone cells
of modern animals, as well as fibrous bone matrix tissue and small,
round red structures resembling red blood cells. The 68-million-year-old
tissue had all the hallmarks of collagen — the main component of
connective tissue and one of the most abundant proteins in living
animals. “When I first saw [the tissues], my first thought was, ‘Those
aren’t supposed to be there.’ Back then, I didn’t think soft-tissue
preservation was possible either,” Schweitzer says.
Credit: K. Cantner, AGI.
As far as biomolecules go, collagen is tough, but nobody thought it
could be that tough. The oldest nondinosaur collagen recovered to date
came from a 3.5-million-year-old camel fossil
found
in the high Arctic of Canada. The unique geochemical conditions
required for long-term preservation of proteins like collagen are still
unknown. In fact, the process of fossilization in general remains
somewhat mysterious, in part because the rigors of geologic time are
impossible to reproduce in the lab. In conventional fossils, just hard
parts such as bones, teeth and shells remain; soft parts are typically
lost to predation or decay before being fossilized.
In exceptional
cases, such as the fossils of the
Burgess Shale in British Columbia,
delicate soft-tissue structures, such as gills or stomach contents, are
mineralized and preserved as rock, but what Schweitzer found was even
stranger: actual soft tissue that had somehow escaped scavengers,
rotting and the ravages of time for 68 million years.
“Living bone is made up of minerals plus organic matter,” Schweitzer
says. After death, as the organics, such as collagen, degrade, they
leave behind voids in the bone at the molecular level. During
fossilization, those voids fill with minerals, which help preserve the
bone for millions of years as a fossil. “The leaving of the organics is
part of what allows the fossil to form,” she says. “But in reality, this
textbook process of fossilization doesn’t explain all the various modes
of fossilization we see, some of which we have no idea about how they
happen.”
Repeatable Results
Many dinosaur fossils have been found in Montana, including the 68-million-year-old
T. rex and an 80-million-year-old
Brachylophosaurus canadensis
in which Schweitzer’s team identified collagen, blood vessels,
hemoglobin, the proteins actin and myosin, and molecules called histones
that help package DNA. Credit: Mary Schweitzer.
Since their initial discovery in 2005, Schweitzer’s team has published
dozens
of studies identifying collagen, blood vessels, hemoglobin, the
proteins actin and myosin, and molecules called histones that help
package DNA, in the 68-million-year-old
T. rex and in an 80-million-year-old hadrosaur, both found in Montana.
When identifying unusual tissues, Schweitzer starts with
high-resolution light microscopy to examine samples for evidence of deep
age: If tissues are actually many millions of years old, certain levels
of damage are expected. “Does it look altered from a living tissue? Is
it possible these are original molecules?” Schweitzer asks. Then, she
turns to antibodies, which recognize and bind to certain types of
proteins present in a sample. “Antibodies are extremely specific. If I
can get a signal, then it justifies the added expense and destruction of
[the sample required for] mass spectrometry sequencing,” she says. Mass
spectrometry is the definitive method used to identify and characterize
both modern and ancient proteins.
The team’s most recent
study, published this year in the Journal of Proteome Research, revisited collagen they recovered from the hadrosaur. In a 2009
study,
Schweitzer’s team identified three fragments, or peptides, from Type 1
collagen. Peptides are made up of amino acids — the building blocks of
protein — and each of the fragments was about 15 amino acids long. As
read by a mass spectrometer, the three fragments appeared to be similar
to collagen 1 peptides found in living reptiles. For the 2017 study, the
team worked with new samples from the same hadrosaur and used updated
extraction methods and more sensitive mass spectrometry to identify
eight peptides: six new and two that matched the 2009 fragments. The new
fragments were found to line up most closely with collagen found in
modern birds — the surviving dinosaurs.
The dinosaur bone at the base of the rock in the center of the image
was found by Schweitzer’s team in Montana. Credit: Mary Schweitzer.
“This latest study is an effort to show that our data are
repeatable,” Schweitzer says. “Everything was different, except for the
source dinosaur. We worked in a different lab, with different
instruments, using different extraction buffers and different analytical
tools, and we still got the same sequences.”
Ideally, other groups would also validate the findings, but not many places are equipped to study such ancient proteins, says
Elena Schroeter,
a postdoctoral researcher at North Carolina State University and lead
author of the 2017 study. “An important step in the scientific process
is replication of data in other labs. That’s been one of our major
problems: This is high-risk research, it’s hard to get funding, and it
is very expensive,” she says. “You need to be able to dedicate a lab
exclusively to ancient proteins to keep everything isolated, to avoid
the possibility of cross-contamination, and not many places are set up
for that.”
The new study is “the most convincing evidence I’ve seen yet that these proteins really are from dinosaurs,” says
Enrico Cappellini,
a professor of paleoproteomics at the Natural History Museum of Denmark
in Copenhagen. “[Schweitzer’s] lab is very careful about
contamination,” he says. “They’re doing everything right in keeping the
ancient stuff totally separate from modern stuff. My inclination is that
their data are authentic, but there’s still room for improvement. I can
understand why many of my colleagues are not yet convinced.”
Bone cell (left) and red blood cells (right). The brown cells (in focus) are surrounded by a white fibrous matrix from the
B. canadensis specimen that Schweitzer’s team studied. Credit: both: courtesy of Mary Schweitzer.
Cappellini says he would like to see the extraction procedure
simplified. Schweitzer’s technique “has a lot of steps, and with each
step, you get a loss of peptides. With a less convoluted procedure, they
might be able to recover a higher number [of peptides] and other labs
might be able to follow the technique,” he says.
One of the most vocal critics has been
Matthew Collins,
a biochemist at the University of York in England who specializes in
recovering ancient proteins. “Mary Schweitzer is either doing something
very right or she’s doing something very wrong,” Collins says. At this
point, the most convincing data would come out of an independent lab, he
says. “I would be really happy if other labs could replicate
[Schweitzer’s] data. [My lab has] tried and we’ve been unsuccessful. So
far, they’re the only ones who have been able to do this.”
Much of the criticism from Collins and others involves questions
about contamination, either from bacterial biofilms that invaded the
fossil long ago and produced the soft-tissue-like materials, or from
modern contaminants introduced in the lab. In May,
Michael Buckley of the University of Manchester in England and his colleagues
published
a study in Proceedings of the Royal Society B that highlights the
difficulties of discriminating dinosaur proteins from modern
cross-contaminants. Buckley and his team extracted collagen from modern
ostrich bone and identified protein sequences identical to those found
by Schweitzer’s team in the hadrosaur in their January study and the
tyrannosaur in the original 2005 study. The findings were “a bit
suspicious,” Cappellini says. They mean “that either 80-million-year-old
dinosaur proteins are identical to those found in modern ostriches or
that [Schweitzer’s] lab was contaminated with ostrich proteins.”
Schweitzer’s team recovered soft tissue from a
B. canadensis fossil found in Montana. Credit: Pedro Salas.
Schweitzer says it’s understandable that some of the hadrosaur
proteins will be identical to those found in modern ostriches — because
collagen is known to be a highly conserved molecule across a diverse
array of species. Dinosaurs and their modern relatives — birds — could
share some of the same protein sequences, she says. “Many molecules are
conserved across species, over millions of years. They work, so they
remain unchanged by evolution.” In fact, she says, some amount of
sequence overlap would be expected, so the similarities between the
hadrosaur and ostrich peptides don’t mean there was contamination in her
team’s samples.
The limited availability of established protein sequences is one of
the big challenges in this field, she says. “We are dealing with the
limitations of an emerging discipline. When you have a
66-million-year-old protein, what do you compare it to?” Currently,
Schweitzer’s team has to settle for comparing protein sequences to those
of extant bracketing species such as birds and crocodiles. “But the
databases are very limited for creatures that are not mammals. These
animals [ostriches and crocodiles] were not represented in existing
databases until we put them there. ”
Preserved by Iron?
Field emission scanning electron microscope images of demineralized ostrich bone matrix (left)
versus B. canadensis bone matrix (right). Both show osteocytes (bone cells). Some of the ostrich proteins are identical to the B. canadensis proteins. Credit: both: courtesy of Mary Schweitzer.
Schweitzer and her colleagues feel they have demonstrated that
contamination isn’t undermining their sensational findings, but they
have not yet figured out how ancient tissues can persist for millions of
years. “That’s the million-dollar question. How on Earth is this
possible?” says
Tim Cleland,
a molecular biologist at the Smithsonian’s Museum Conservation
Institute in Washington, D.C., who has co-authored papers with
Schweitzer, including the 2017 study.
“I have yet to hear a plausible explanation for how soft tissues can
be preserved for this long,” Collins says, citing the huge leap of time
from 3.5 million years to more than 66 million years. “The more I
understand about it, the more difficult it is to believe it can happen,”
he says. “There may be something very special about these fossils, but
for me they’re defying basic chemistry and physics. I’d like to see
better comprehension of what happens when proteins decay and how decay
might be suspended indefinitely.”
In 2013, Schweitzer’s team offered a
hypothesis:
The preservative could be iron. Tissues from both the tyrannosaur and
the hadrosaur they studied were found to be chock-full of iron
nanoparticles. Iron is abundant in living organisms, especially in the
blood, where it makes up part of the oxygen-transport protein
hemoglobin. Iron is notoriously bioreactive, so much so that organisms
must take biochemical precautions to prevent excess iron from causing
tissue damage. But while iron can be detrimental to living tissues, it
can act like a preservative in dead tissues, Schweitzer says.
“When a dinosaur dies, it has a boatload of iron in its blood and
muscles. And when that iron is released into the carcass, it goes about
cross-linking like crazy, tying proteins and cell membranes in knots,
basically acting a lot like formaldehyde,” she says. Tissues treated
with formaldehyde can sit in a jar on a shelf for decades, but decay is
only delayed, not arrested. To demonstrate iron’s preservative
capabilities, Schweitzer extracted blood vessels from modern ostrich
bones and treated them with iron-rich hemoglobin. “The control [samples
turned to] mush in three days, but the vessels exposed to hemoglobin are
still sitting on a shelf in my lab at room temperature six years
later,” she says. Collins says he remains skeptical. “Iron may slow down
the decay process but it’s not clear how it could be arrested
altogether,” he says.
Finding and Storing Soft Tissue
The internal conditions of a carcass play a role in the initial stages
of tissue preservation, but the external environment is crucial for
fossilization. “Every fossil has its own unique geochemical
environment,” Schroeter says. And while paleontologists have a good idea
where they should look for dinosaur fossils — in Mesozoic sedimentary
rocks, usually in hot, dry places such as eastern Montana and
northeastern Utah — they’re still figuring out what conditions are more
likely to support fossils with preserved soft tissues and how much might
exist. “It’s an open question, how much of this [soft-tissue] material
is out there,” Cleland says. Soft tissue in dinosaur fossils is rarely
found, but that may be because “people rarely look” for it, Schroeter
says. Credit: K. Cantner, AGI; inset figures modified from Schweitzer et
al., Proceedings of the Royal Academy B, 2006.
It may be more common than we realize, Schweitzer adds. Her team has
found evidence for soft tissue in about a third of the several dozen
specimens they have studied. “We will probably find more if we look
more,” she says.
Schweitzer’s team has been working on formulating a profile of
burial environments that seem to be conducive to protein preservation.
“Fossils that are found in sandstone seem to have better preservation
than those that come from shale or claystone, but we don’t really know
why that is yet,” Cleland says. “The only thing we know for sure is that
the carcass needs to be buried very quickly after death.” The dinosaurs
that Schweitzer’s team has worked with have been found partially
articulated — a sign that the carcasses were not pulled apart by
scavengers.
“You have to be pretty lucky to find a well-preserved dinosaur bone,
let alone a whole dinosaur,” Schroeter says. “There’s a huge range of
preservation out there. Some fossils have perfect histological structure
and look just like pristine bone in terms of their microstructure,
while other fossils look just like rocks.”
Once fossilized materials are removed from the ground, whatever
equilibrium they were in can be disrupted. In an effort to preserve any
remaining soft tissues and prevent contamination, Schweitzer’s team has
developed a new approach to excavating and handling fossils. “It’s best
to keep the fossils in the sediment they were in until you get them into
a controlled lab setting,” Schweitzer says, even though this sometimes
results in massively jacketed fossils that are a logistical nightmare to
transport. “We can remove the fossil wearing gloves, without using any
glues, using sterile instruments and containers in an isolated lab.”
To preserve soft tissue and prevent contamination, Schweitzer’s team
wraps dinosaur fossils in massive plaster jackets and transports them —
however cumbersome they are — back to the lab, where researchers can
remove the fossil using sterile gloves, instruments and containers.
Credit: Ken Lacovara.
Then, instead of being stored in museum drawers where they might be
contaminated, the fossils are kept in sterile glass jars, in special
cabinets isolated from other fossils or modern samples, in a room far
removed from other specimens. The team is also developing better
techniques for storing these samples long term. “Technology gets better
and more sensitive by leaps and bounds all the time,” Cleland says. “We
want these samples to be around in [a decade] and even longer.”
Constructive Results From Destructive Sampling
Testing for proteins requires destructive sampling, in which bits of
the fossils are inevitably destroyed. “Plenty of curators aren’t big
fans of destructive sampling,” Schroeter says. “I’d really like to test
more specimens, but it can be hard to get people to let you grind up
their fossils.”
Paleontology has long relied on scant clues gleaned from bones
turned to stone, and the possibility of introducing molecular techniques
into the field is tantalizing to some researchers. “Part of the power
of molecular paleontology is the ability to look at dinosaurs from a
different angle,” Schroeter says. “Morphology can get you pretty far,
but there are a lot of things that bones can’t tell you.”
Even skeptics agree that molecular paleontology has great potential.
“If soft-tissue preservation is possible, it would open vast areas of
the fossil record to molecular analysis,” Collins says. “Honestly, I
really hope [Schweitzer] is right.”
Many scientists are loathe to allow even tiny pieces of fossil, like this piece of rib from a
Lufengosaurus, a sauropod, to be destroyed — a necessary process for molecular paleontology. Credit: Lee et al., Nature Communications, 2017.
A molecular approach could answer long-standing questions about the
physiology of dinosaurs, such as how they regulated body temperature,
how their cardiovascular systems were able to support such large
creatures, and how they grew and reproduced, as well as resolve how
various species were related to one another, how they spread around the
planet, and how some lines evolved into birds. But the field has a long
way to go.
One of the biggest hurdles is the difficulty of proving endogeneity —
that the proteins recovered come from a dinosaur and not a bacterial or
modern source (see sidebar,
page 45).
“When you are testing fossils, it is critical to use multiple methods
to examine and reexamine, test and retest different aspects of the
molecules we think we’re recovering,” Schweitzer says. “To get the field
moving forward, we need to come to a consensus on which methods are the
most efficient, and which methods can tell us the most with the least
amount of [sample] destruction.”
Other teams have developed methods of testing for ancient preserved
proteins that don’t require destructive sampling, but the methods’
reliability is uncertain and they provide limited information about
samples. For example,
Yao-Chang Lee of the National Synchrotron Radiation Research Center and National Central University in Taiwan and colleagues published a
study in Nature Communications this year in which they claim to have recovered collagen from a 195-million-year-old long-necked
Lufengosaurus.
The researchers described using nondestructive infrared spectroscopy to
identify the protein. But there’s a flaw with this method, Cappellini
says. “With this technique, you can see a chemical signature of what
might be a protein, but it’s not a clear fingerprint like you get with
mass spectrometry.” And that’s just not good enough, he says. “On
controversial samples this ancient, it’s necessary to have
unquestionable evidence.”
The Future Could Be Molecular
Recep Avci (standing) and Jahson Suo look at images of soft tissue from
dinosaurs under a microscope. Credit: Kelly Gorham, Montana State
University.
After weathering years of
controversy, Schroeter says she is beginning to feel like a mouse
running endlessly on a wheel: “Some of us in Mary’s lab feel like we’re
stuck continuously arguing that these proteins can persist, even though
we’ve shown repeatedly that they do,” she says. “We want to get beyond
these same arguments and focus on what we can learn from these tissues.”
Schweitzer says she chooses to respond to her critics by working
harder. “The doubters push me to do everything the best I can do it,”
she says. “I want to make this work last for the ages.” Neither
Schweitzer nor Schroeter are holding their breath for a single piece of
evidence that will satisfy their critics once and for all. “When making a
scientific argument ... you want a bunch of different lines of evidence
that all point toward the same thing,” Schroeter says.
Whether it’s finding blood vessels from hadrosaurs, like this
Saurolophus (right), or determining the colors of feathers on dinosaurs like the
Jiangxisaurus
(left), researchers hope molecular paleontology can reveal many
long-kept dinosaur secrets. Credit: right: L. Xing and Y. Liu, PLOS ONE,
CC BY 2.5; above: Danny Cicchetti, CC BY 3.0.
Interest in the field is growing, but the number of labs set up for
this kind of work remains limited, Schroeter says. “Paleontology has
always been a little slow on the uptake in adopting new technology, but
as more young people get interested, they’ll bring their tech savvy,”
she says. “To be competitive, you have to constantly be thinking of new
questions, new approaches, new ideas. This is how we’re going to raise
the bar for paleontology.”
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