Archaeology/paleontology (“ancient DNA” analysis) Conservation biology Forensic science Taxonomy Power & pitfalls of PCR (polymerase chain reaction) Rapid amplification of specific genomic regions using very 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, 15 o C … take about 100,000 years for hydrolytic damage to destroy all DNA…. Some environmental conditions could extend this time limit…”

Marchant Nature 472: 404, malaria parasite (Plasmodium) identified as possible cause of death - used 3,300 year-old DNA (bone) from King Tutankhamun’s tomb to construct a 5-generation family tree Hawass J. Amer.Med.Assoc. 303:638, 2010 Stoneking Nature Rev Genet 12:603, 2011 Comparison of 3 high-throughput methods of sequencing ancient DNA

“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 Dispersal of modern human populations “Out-of-Africa mitochondrial Eve” hypothesis... Y chromosome data are also in agreement Stoneking Nature Rev Genet 12:603, 2011

Are Neanderthals the direct ancestors of modern Europeans? First modern humans appeared ~ 200,000 years ago First known Neanderthals ~ 300,000 years ago Neanderthal skeleton found in Germany (~ 30,000 – 100,000 years old) 1997 – S. Paabo – PCR amplified & sequenced mitochondrial DNA (D-loop region, 379 bp) from skull and compared to 986 living 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 genetic lineages to common ancestor when traced 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 myosin multi-gene family) Stedman Nature 428:415, 2004

Compared rates of non-synonymous vs. synonymous nt substitution to assess degree of functional constraint Stedman Nature 428:415, 2004