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Section VI: Section VI: PaleomolecularPaleomolecular biologybiology

Topic 1: Ancient DNA

Topic 2: Paleomolecular biochemistry

Ancient DNA: (1) Samples of DNA retrieved from museum specimens, archeological finds, fossil remains, or other non-living sources of historical DNA. (2) The scientific discipline devoted to using ancient DNA to study the process and outcome of biological evolution.

Section VI: Section VI: PaleomolecularPaleomolecular biologybiology

Paleomolecular biochemistry: the scientific discipline devoted to the “resurrection” of an ancestral protein for the purpose of studying how its biophysical properties evolved, or to make inferences about the evolution of the organisms that expressed the protein.

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Ancient DNA: allows us to work with data below the tips

Ancient DNA: can’t do it without PCR

Polymerase chain reaction (PCR): an enzyme based technique for amplifying (making many copies) a specific segment of DNA (template). The technique is amazingly sensitive, huge quantities of DNA can be amplified from extremely small amounts of template DNA.

Before PCR:

• ancient DNA was cloned to make more copies.

• cloning requires much more starting DNA than PCR

• successful cloning of ancient DNA was not reproducible

quagga ancient Egyptian

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Technique invented by Kary Mullis (1983); Nobel price in (1993)


single molecule of DNA ⇒ over 100 billion copies

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Ancient DNA: The power of PCR leads to pitfalls

After PCR:

• need only a single copy of ancient DNA

• PCR can be repeated to satisfy “repeatability” criterion

• trace amounts of exogenous DNA become a serious problem

Due to preservation conditions, many samples will have no DNA:

• even 1 molecule of exogenous DNA could lead to 100 billion copies!

• contamination is the single most serious concern in the study of ancient DNA.

Ancient DNA: contamination with exogenous DNA

Neandertal example:

• remains from 24 locations

• only 4 samples were preserved well enough for DNA preservation

• PCR applied to all samples with primers for a human gene:

• Most samples yielded DNA sequences.

• Most DNA must have been human contamination.

Human DNA is almost ubiquitous in specimens and laboratory environments!

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Ancient DNA: The first “Dino-DNA” is reported in 1994

Note: No phylogenetic analyses were preformed. The authors argued that phylogenies from such short sequences were unreliable

Ancient DNA: a spectacular bubble is burst in 1995

Two different phylogenetic analyses reveal:

• bone DNA can’t be 80 million years old

• bone DNA probably a human contaminant

The bone DNA was probably a copy of the human cyt-bgene that was translocated to the nuclear genome of humans and became a pseudo-gene. Hence that sequence was different from all other “functional” cyt b genes in the database, but was phylogenetically closest to humans.

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Ancient DNA: contamination with exogenous DNA

Following the “Dino-DNA” incident, claims of DNA from amber-entombed insects also were reported to be unreliable. The area of ancient DNA research had suffered a very public fall from grace.

Ancient DNA renaissance:

1. Dedicated ancient DNA facilities

2. Rigorous authenticity criteria

Ancient DNA: dedicated ancient DNA facilities

Workspace:

• isolated from all other molecular biology

• regularly cleaned with bleach

• regularly irradiated with UV

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Ancient DNA: rigorous authenticity criteria

2. Negative controls: (i) extraction blanks, and (ii) negative PCR controls

3. Multiple PCRs from the same tissue extract, repeated over several extracts

4. Verify inverse correlation between PRC efficiency and length of template.

5. Biochemical assessment of likelihood of DNA preservation.

1. Clone and sequence multiple positive PCR products

6. Exclude possibility that mtPCR product is not a nuclear insertion.

7. Design PRC strategy from contig assembly from multiple, overlapping fragments.

8. Independent replication in a second laboratory at another location.

8. Employ species specific primers whenever possible

Criteria for establishing authenticity of ancient DNA

Criteria are continuously being added to this list.

In the case of ancient DNA from “modern humans” it is impossible to establish authenticity, even by the above criteria.

Ancient DNA: all DNA is easily damaged

Living cells: continuously repaired by enzymatic processes

After Death:

• repair process shuts down

• other destructive enzymes rapidly break down DNA

• normally sequestered in certain cellular compartments

• e.g., lysosomal nucleases

• organisms rapidly feed on and degrade DNA

• bacteria

• fungi

• insects

Only rarely are conditions (cold and dry) suitable for DNA preservation

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Ancient DNA: molecular damage

When you find ancient DNA:

Very little DNA

Very short (100-500bp) segments

Limits the efficiency of PCR

Prevents PCR

Results in misincorporationsduring PCR

Big problem is it occurs early in the PCR process

Ancient DNA: molecular damage

DNA damage accumulates progressively:

• relentless and irreversible

• stops only when all nucleotide sequence is lost

• all DNA sequences degrade; its only a matter of time

Only rarely are conditions suitable for DNA preservation

Warm, wet and old:

• fast degradation

• ancient DNA? ⎯forget it!

Cold, dry and recent:

• slower degradation

• ancient DNA? ⎯rarely

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Ancient DNA: What is achievable.

32 - 47,000 YBPBurger et al. 2004Cave lion

13,000 YBPHoss et al. 1996Giant ground sloth

~3300 YBPCooper et al. 1992Moas

~200 YBPThomas et al. 1989Tasmanian wolf

140 YBPHiguchi et al. 1984Quagga Museum skins

Hofreiter et al. 2002

Hagelberg et al. 1994

Krings et al. 1997

Paxinos et al. 2002

Adcock et al. 2001

Paabo et al. 1989

Reference

49,000 YBP Cave bear

Mammoth

Neandertal humans

Hawaiian geese

Ancient modern humans

Recent Modern humans (Bog bodies)

Organism

8-15,000 YBP

> 16,000 YBP

> 30,000 YBP

7500 YBP

47,000 YBP

Age of DNA

Selected examples of authentic ancient DNA

The deepest authenticated DNA dates to the late Pleistocene (50,000 years BP)

Ancient DNA: What is achievable.

Many of the major natural history museums now have ancient DNA labs and protocols in place

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Ancient DNA: What is NOT achievable?

135 myaDeSalle et. al. (1993)Insects entombed in amber

Vreeland et al. (2000)

Cano & Borucki (1995)

Woodward et al. (1994)

Goldberg et al. (1990)

Reference

Bacteria in salt crystals

Gut bacteria in insects entombed in amber

Dinosaur bone

Plant compression fossils

Organism

17-20 mya

80mya

25-40 mya

250 mya

Age of DNA

Examples of spectacular reports of ancient DNA

Given rate of decay: DNA not expected to survive more than 1 million years

Consensus opinion: DNA > 1 million years is an artifact.

Ancient DNA: applications

1. Relationships of extinct species

2. Verifying fossil taxonomy

3. Population history and biogeography

4. Estimating the rate of evolution

5. Inferring diet and behavior

6. Medical archeology

7. Origins of domestication and agriculture

8. Human evolution

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Ancient DNA: Are New Zealand's flightless birds monophyletic?

Moa: large flightless bird of New Zealand.

• Went extinct in modern times; probability 300-400 hundred years ago.

• Moas were present in New Zealand when it was colonized by Maori people

• Moari regularly ate Moas; brought dogs and rats; probably important part of extinction

• oldest Moa bones: 2 million years

Kiwi (3 species): last surviving lineage (in N.Z.) of the order of flightless birds (ratites) that included the Moas and colonized New Zealand

Moas and Kiwis assumed to be closely related, probably sister taxa

Ancient DNA: Are New Zealand's flightless birds monophyletic?

Ratites, 10 living species:

• Ostrich (1 sp: Africa and formerly Asia)

• Emu (1 sp: Australia)

• Cassowaries (3 sp: Australia and New Guinea)

• Rheas (2 sp: South America)

• Kiwis (3 sp: New Zealand)

Ancient DNA:

• 4 “species” of Moa

• 400 bp 12srRNA gene

• DNA from bone and soft tissue from museum collections

Possibly 11 species, all extinct

New Zealand and Australia separated about 80 mya, Moa fossils go back 2 million years

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Madagascar: Another island with giant flightless birds

Madagascar elephant bird:

• Aepyornis maximus (right)

• 2 other species

• extinct

Ancient DNA: What about the elephant bird?

Ancient DNA:

• Complete mtDNA genomes for 2 “species” of Moa

• 1000bp for extinct Madagascar “elephant bird”

Extant sp: long range PCR

Extinct species: PCR in bits and bobs

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Ancient DNA: species phylogeny of ratites

• Confirm that Moa and Kiwi are not monophyletic

• Elephant bird and Ostrich not monophyletic!

• 80 mya land bridges connected much of the southern hemisphere

• A flightless ancestor could have dispersed at that time

13 mya82 mya

65-72 mya

Ancient DNA: verify the fossil based taxonomy

Possibly 11 species, all extinct

Morphology: 3 species (1,2,3 below)

Ancient DNA:

Morpho-species 1: All males

Morpho-species 2 and 3 all females

Haddrath and Baker (2001)

Ancient DNA data study of 3 species:

• mtDNA for phylogeny

• nucDNA to determine sexmtDNA tree:

• Three species not monophyletic

• Probably 1 species with population subdivision.

• Species reduced:11⇒ 9

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Ancient DNA: species phylogeny of extinct animals

Anteater

Naked-tail Armadillo

3-toed sloth

2-toed sloth

Nine-band Armadillo

Anteater

Naked-tail Armadillo

3-toed sloth

2-toed sloth

Nine-band Armadillo

Marsupial wolf: Thylacinuscynocephalus

Morphology: some characters suggest relationship with South American marsupials

Ancient DNA: relationship with Australian marsupials

Adelie penguin colonies are ideal for this type of study:

• Nest in distinct colonies

• Colonies in cold and ice-free areas

• High density and mortality in colonies.

• Large deposits in a cold and dry environment

• Sub-fossil deposits go back 7786 years

• Best preserved DNA discovered to date

Ancient DNA: Estimating the rate of molecular evolution

Traditional methods of estimating evolutionary rates per unit time:

Indirect: number of substitution between a pair of taxa, usually under a clock

Direct: mutation rates per generation

New method uses ancient DNA:

Adelie penguin

Pygoscelis adeliae

Adelie penguin

Pygoscelis adeliae

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Ancient DNA: Estimating the rate of molecular evolution

Mitochondrial control region DNA (HVRI)

Extant:

• 380 living bird blood samples

Ancient:

• 96 bone samples

• Each with a date; range: 88 to 6424 ybp

Estimated rate:

• Method integrates over uncertainty in network [e.g., B]

• 0.4-1.4 substitutions/site/Myr

• 2 – 7 times faster than indirect estimates [0.2s/s/Myr]

• supports a nearly neutral model of evolution for mtDNA

Ancient DNA: population history of ice age brown bears

Brown bear

Ursus arctos

Brown bears:

• Extensive holarctic distribution (Europe, Asia, North America)

• Highly structured populations

• Entered north America via Beringia , perhaps 60-70,000 ybp

• Spreading-out only 13,000 ybp

• Presently there are three-four genetically distinct groups

• Well preserved ice age bears in perma-frost

Genetic groups of brown bears:

Group II

Group IIIa

Group IIIb

Group IV

Dynamic Holarctic history:

• Intercontinental Migrations

• Glaciations

• Local extinctions

• Brown bear distribution changed dramatically

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Radiocarbon DatesPhylogeny of extant and ancient bear DNA

Ancient mtDNA:

• 36 bone samples

• 10-50000 ybp

Results:

• Modern genetic structure present in bear populations 30-40,000 ybp

• Little change since then (except loss of group IIIc)

• Ancient populations were genetically diverse

Grizzly bear

Polar bear

Short-faced bear

A new character: Short-faced bear (Arctodus simus)

The short-faced bear is a hyper-carnivore

3m

[9.8ft]

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Brown bears disappear

• Extinct in east Beringia

• no fossils at all

Short faced bear present

• Much larger

• Hypercarnivorous

Brown bears return

• Ancestral diversity is present

Short faced bears go extinct as brown bears return

Ancestral Brown bears

Possess all of modern diversity

Grey shading indicates extent of glaciations

Large dashed line indicates migration boundaries

Small dashed line indicates coastline


Substantial levels of ancestral diversity have been maintained; surviving local extinctions, re-colonization, and extreme competition with short-faced bear.

This study changed our view of modern brown bear populations:• The modern geographic distribution of genetic lineages co-existed in the past

• The modern genetic lineages did not evolve in their current environments

• The modern genetic lineages of bears cannot be subspecies adapted to different local environments [as has been suggested]

• Management plans that maintain this structure and try to prevent interbreeding are misguided.

• Modern Brown bear populations do not reflect the long term history of the species

Important implications for conservation genetics

Brown bear

Ursus arctos

Brown bear

Ursus arctos

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Ancient DNA: molecular medical archeology

Molecular medical archeology: discipline of science devoted to retrieval of ancient DNA sequences for the purposes of studying pathogenic organisms and their roles in human populations

Examples include:

• How do the pathogens of the great epidemic of 1918 differ from today’s pathogens?

• What was the precise culprit of the black death?

• Did tuberculosis exist in the New World before the arrival of Europeans?

• What was malaria’s role in the fall of Rome?

Very cool stuff!

You need to understand population genetics!

You need to understand molecular evolution!

Ancient DNA: Tuberculosis in ancient Egypt

Ancient DNA:

• Insertion sequence unique to Mycobacterium tuberculosis

• 37 humans from the “tombs of nobles” from the necropolis of the city of Thebes

• remains as old as 3000 BCE

• only females worked on male samples and vice-versa

Historical background:

• Tombs hold social upper class during “Middle” and “New Kingdom” period

• Most prosperous time in ancient Egyptian history

• Thebes was capital of empire at this time

• Conditions were good

• Population about 10,000 during this period

• Dense crowding during this period

Tuberculosis found in:

• c. 40% of non-specific cases

• c. 15% of completely unremarkable bones!

• Tuberculosis seems to be significantly higher than previously thought!

• Thebe’s population known for unusually low life expectancy!

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