Archaeology/paleontology (“ancient DNA” analysis)

Conservation biology

Forensic science

Taxonomy

Power & pitfalls of PCR (polymerase chain reaction)

Rapid amplification of specific genomic regions usingvery small amounts of DNA

But … damaged DNA may lead to sequence artifacts

or “ancient” DNA may be contaminated with “modern” DNA

APPLICATIONS OF MOLECULAR PHYLOGENETICS

Forms of DNA damage likely to affect ancient DNA

8 clones of PCR-amplified mtDNA from 26,000 year old cave-bear bone

C/G to T/A changes due to deamination of C residues?

Hofreiter, Nat.Rev.Genet. 2:353, 2001

“ … to consider amplification of DNA molecules older than one million years of age is overly optimistic.” (Svante Paabo lab)

“Note that direct sequencing would lead to ambiguous results at least at two positions (arrows)”

“Assuming neutral pH, 15oC … take about 100,000 years for hydrolytic damage to destroy all DNA…. Some environmental conditions could extend this time limit…”

“Tasmanian wolf” classified as relative of South American marsupials (based onmorphology)

… but PCR of mtDNA from museum specimen …

Fig. 5.35

“For the first time, the sequence of a near-complete nuclear genome has been obtained from the tissue of an ancient human. It comes from permafrost-preserved hair, about 4,000 years old, of a male palaeo-Eskimo of the Saqqaq culture, the earliest known settlers in Greenland.”

Analysis of DNA from an ancient human - Nature Feb. 11, 2010

“We identified 353,151 high-confidence single-nucleotide polymorphisms (SNPs), of which 6.8% have not been reported previously.”

Rasmussen et al. Nature 463:757, 2010

Since some SNPs are reliable markers for known phenotypes of individual humans...

... could predict that Saqqaq man had brown eyes, non-white skin, thick dark hair, dry ear wax and increased susceptibility to baldness

Lambert Nature 463:757, 2010

Comparison of Saqqaq high-confidence SNPs to those of contemporary human populations

- support for migration from Siberia into New World ~ 5,500 years ago, independent of that giving rise to modern Native Americans and Inuit.

Rasmussen et al. Nature 463:757, 2010

“Principal component analysis to capture genetic variation”

Hartl & Jones Fig. 14.30

Dispersal of modern human populations

“Out-of-Africa mitochondrial Eve” hypothesis

Are Neanderthals the direct ancestors of modern Europeans?

First modern humans appeared ~ 200,000 years ago

First known Neanderthals ~ 300,000 years ago

1856 - Neanderthal skeleton found in Germany (~ 30,000 – 100,000 years old)

1997 – S. Paabo – PCR amplified & sequenced mitochondrialDNA (D-loop region, 379 bp) from skull and compared to 986living humans

~ 3 x more differences between human-Neanderthal than human-human

and no particular similarity between Neanderthal-European human

Krings, Cell 90: 19, 1997

If lineages diverged ~ 600,000 years ago,

… suggests that Neanderthals were evolutionary dead-end

(ie. branch that became extinct without any direct genetic contribution to modern human lineage)

Science 277: 176, 1997

Coalescence times – joining of geneticlineages to common ancestor whentraced back in time

2000 – mtDNA of Neanderthals from Croatia & the Caucasus analyzed

Hofreiter, Nat.Rev.Genet. 2:353, 2001

“A reanalysis of the ancient mitochondrial DNA”... Gutierrez, Mol Biol Evol 19: 1359, 2002

High variation in substitution rate within D-loop region

- different outcome with HVI vs. HVI + HVII sequences

HVI part of D-loop

HVI + HVII

Previous sequence data

Figure 5.28

Chimpanzee outgroup too distant? “long branches attract” phenomenon

p.214

Science 314: 1071, 2006

Sequence analysis of Neanderthal genome (Nov. 2006)

Stedman Nature 428:415, 2004

MYH16 (member of myosinmulti-gene family)

Stedman Nature 428:415, 2004

Compared rates of non-synonymous vs. synonymous nt substitutionto assess degree of functional constraint

Stedman Nature 428:415, 2004