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

Nature,1993 - but in fact this turned out to be contaminating “modern” DNA In some cases, mistakes were due to organellar DNA fragments (pseudogenes) integrated into “modern” nuclear genome

Ancient DNA studies (1984 – 2004) Willerslev & Cooper Proc Roy Soc 272:3, 2005

Dispersal of modern human populations Hartl & Jones Fig. 14.30 Stoneking Nature Rev Genet 12:603, 2011 “Out-of-Africa mitochondrial Eve” hypothesis ... Y chromosome data are also in agreement

Relationship between Neanderthals and “modern humans”? First modern humans appeared ~ 200,000 years ago Our closest known relatives are Neanderthals who lived from ~ 600,000 to ~30,000 years ago 1997 – S. Paabo – PCR amplified & sequenced mitochondrial DNA (D-loop region, 379 bp) from skull and compared to 986 living humans Krings, Cell 90: 19, 1997 (from skeleton discovered in cave in Germany in 1856) ~ 3 x more differences between human-Neanderthal than human-human and no particular similarity between Neanderthal-European human 2000 – mtDNA of Neanderthals from Croatia & the Caucasian Mountains region also analyzed Estimate of divergence time: 550,000 – 690,000 yr (using chimpanzee as outgroup) … suggesting that Neanderthals were evolutionary dead-end Hofreiter, Nat.Rev.Genet. 2:353, 2001 (ie. branch that became extinct without any direct genetic contribution to modern human lineage)

2002 “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

2006 – two studies report 1 million bp from Neanderthal nuclear genome Science 314: 1071, 2006 2007

2008 – first complete Neanderthal mitochondrial genome (16 kb) 2007 – nuclear gene for pigmentation suggests Neanderthals had red hair Neanderthals ... They're Just Like Us? National Geographic 2008 – first complete Neanderthal mitochondrial genome (16 kb) 2010 – draft sequence of Neanderthal nuclear genome (> 4 billion bp from 3 individuals !!) New rapid sequencing techniques 2011 – mitochondrial DNA analysis from Neanderthal family of 12 individuals dated to ~ 49,000 years ago Providing anthropological insights into kinship structure, mating behaviour…

Inferences about evolutionary relationships from genomic data Stoneking Nature Rev Genet 12:603, 2011 “The genomes provide evidence of gene flow between the three lineages after anatomically modern humans left Africa, drastically changing our view of human evolution.” “Red arrows mark genetic evidence of interbreeding among different populations. Black arrows mark suggested or possible additional gene flow.” Lalueza-Fox & Gilbert Curr Biol 21:R1002, 2011

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