APPLICATIONS OF MOLECULAR PHYLOGENETICS 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

Hofreiter, Nat.Rev.Genet. 2:353, 2001 8 clones of PCR-amplified mtDNA from 26,000 year old cave-bear bone “Note that direct sequencing would lead to ambiguous results at least at two positions (arrows)” Forms of DNA damage likely to affect ancient DNA C/G to T/A changes due to deamination of C residues? “Assuming neutral pH, 15oC … take about 100,000 years for hydrolytic damage to destroy all DNA…. Some environmental conditions could extend this time limit…” “ … to consider amplification of DNA molecules older than one million years of age is overly optimistic.” (Svante Paabo lab) Hofreiter, Nat.Rev.Genet. 2:353, 2001

“Tasmanian wolf” classified as relative of South American marsupials (based on morphology) … but PCR of mtDNA from museum specimen … Fig. 5.35

Analysis of DNA from an ancient human - Nature Feb. 11, 2010 “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.” “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 “Principal component analysis to capture genetic variation” - 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

Dispersal of modern human populations “Out-of-Africa mitochondrial Eve” hypothesis Hartl & Jones Fig. 14.30

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) Krings, Cell 90: 19, 1997 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

If lineages diverged ~ 600,000 years ago, Science 277: 176, 1997 Coalescence times – joining of genetic lineages to common ancestor when traced back in time 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)

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 HVI part of D-loop HVI + HVII Previous sequence data High variation in substitution rate within D-loop region - different outcome with HVI vs. HVI + HVII sequences

Chimpanzee outgroup too distant? “long branches attract” phenomenon Figure 5.28

Sequence analysis of Neanderthal genome (Nov. 2006) Science 314: 1071, 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