Section VI: Section VI: Paleomolecular Paleomolecular biology biology Topic 1: Ancient DNA Topic 2: Paleomolecular biochemistry Section VI: Paleomolecular Section VI: Paleomolecular biology biology 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. 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. 1
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 2
Ancient DNA: can’t do it without PCR Technique invented by Kary Mullis (1983); Nobel price in (1993) Ancient DNA: can’t do it without PCR single molecule of DNA ⇒ over 100 billion copies 3
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! 4
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-b gene 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. 5
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 6
Ancient DNA: rigorous authenticity criteria Criteria for establishing authenticity of ancient DNA 1. Clone and sequence multiple positive PCR products 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. 6. Exclude possibility that mtPCR product is not a nuclear insertion. 7. Design PRC strategy from contig assembly from multiple, overlapping fragments. 8. Employ species specific primers whenever possible 8. Independent replication in a second laboratory at another location. 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 7
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 misincorporations during 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: Cold, dry and recent: • fast degradation • slower degradation • ancient DNA? ⎯ forget it! • ancient DNA? ⎯ rarely 8
Ancient DNA: What is achievable. Selected examples of authentic ancient DNA Organism Reference Age of DNA Quagga Museum skins Higuchi et al. 1984 140 YBP Tasmanian wolf Thomas et al. 1989 ~200 YBP Moas Cooper et al. 1992 ~3300 YBP Recent Modern humans (Bog bodies) Paabo et al. 1989 7500 YBP Ancient modern humans Adcock et al. 2001 8-15,000 YBP Giant ground sloth Hoss et al. 1996 13,000 YBP Hawaiian geese Paxinos et al. 2002 > 16,000 YBP Neandertal humans Krings et al. 1997 > 30,000 YBP Cave lion Burger et al. 2004 32 - 47,000 YBP Mammoth Hagelberg et al. 1994 47,000 YBP Cave bear Hofreiter et al. 2002 49,000 YBP 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 9
Ancient DNA: What is NOT achievable? Examples of spectacular reports of ancient DNA Organism Reference Age of DNA Plant compression fossils Goldberg et al. (1990) 17-20 mya Dinosaur bone Woodward et al. (1994) 80mya Gut bacteria in insects entombed in amber Cano & Borucki (1995) 25-40 mya Insects entombed in amber DeSalle et. al. (1993) 135 mya Bacteria in salt crystals Vreeland et al. (2000) 250 mya 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 10
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? Ancient DNA: • 4 “species” of Moa • 400 bp 12srRNA gene • DNA from bone and soft tissue from museum collections Possibly 11 species, all extinct 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) New Zealand and Australia separated about 80 mya, Moa fossils go back 2 million years 11
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 12
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 82 mya 13 mya 65-72 mya Ancient DNA: verify the fossil based taxonomy Morphology: 3 species (1,2,3 below) Ancient DNA: Morpho-species 1: All males Morpho-species 2 and 3 all females Possibly 11 species, all extinct Haddrath and Baker (2001) Ancient DNA data study of 3 species: • mtDNA for phylogeny mtDNA tree: • nucDNA to determine sex • Three species not monophyletic • Probably 1 species with population subdivision. • Species reduced:11 ⇒ 9 13
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