Department of Evolutionary Biology

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Ancient DNA in Sediments

• Department of Evolutionary Biology
• Zoological Institute
• University of Copenhagen

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Ancient DNA Studies
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DNA from Sediments
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Sample Information
Samples Site Age range (B.P.)
Permafrost     
1/02/0.5 Kolyma lowland, Plakhin Jar modern tundra soil
1/93/4.0 Kolyma lowland, Kon'kovaya river 10.42545 yr
2/01/4.8 Laptev Sea coast, Cape Bykovskii 18.98070 yr (8-9 kyr)
7/90/1.6 Kolyma lowland, Chukochia river 20-30 kyr
3/01/20.7 Laptev Sea coast, cape Svyatoi Nos 300-400 kyr
4/01/9.2 Laptev Sea coast, cape Svyatoi Nos 300-400 kyr
6/90/30.7 Kolyma lowland, Chukochia river 1.5-2.0 Ma
6/90/31.1 Kolyma lowland, Chukochia river 1.5-2.0 Ma
1/99/14.5 Beacon Valley, Antarctica 8.1 Ma
New Zealand    
Cave sediment Clutha River 62450 yr
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Microscopy

• Cells in the bacterial size range (about
  107cells/ gww, average cell volume 0.03-0.05
  µm3/cell)
• Occasional fine rootlets (2 mm in diameter),
  seeds and small unidentifiable multicellular
  fragments
• No bone/hair/identifiable animal soft tissue

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PCR Based Analyses

• 4 x 0.25gww soil
• FAST PREP
• DNA extraction/purification
• PCR (universal/specific primers for
  rbcL/mtDNA)
• Cloning
• Sequencing
• BLAST (GenBank)/phylogenetic analysis

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Precaution, Controls, Criteria

• Special rotation-column coring method
• Spiking with bacterial Serratia marcescens
• Isolated, dedicated clean lab.
• Isolated ventilation system, UV-radiation, flow
  hood
• Facemasks, gamma-sterilized glows, hats
• Removal of core surfaces
• Cleaning of reagents/tools UV, HCL, bleach,
  ultrafiltration
• Extraction/ PCR controls
• Cloning
• Independent reproducibility of results
• Phylogenetic criteria

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Important!

• Not previously worked with in the Copenhagen
  lab (at that stage)
• plant rbcL DNA
• DNA from Arctic or NZ animals (including
  megafauna) except for Reindeer mtDNA
• Previously produced PCR products is a major
  source of contamination

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Amplification Results

• Plants (rbcL about 130 bp)
• PCR products up to 300-400 kyr (including NZ
  cave site)
• No PCR products million year old samples
• Animal (mtDNA 88-234 bp)
• PCR products up to 20-30 kyr (including NZ cave
  site, only primers for bird mtDNA)
• no PCR products 300-400 kyr and million year old
  samples
• The results were independently confirmed in
  Oxford

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Plant identifications (multiple GenBank
sequences showing gt96 similarity to the clones
reproducibility confirmed by a bootstrap test )
Class or Sub-class ?9 Order ?22 Order ?22 Family ?28 Family ?28
Liliopsida  Coniferopsida Asteridae  Rosidae CaryophyllidaeEudicotyledon Bryidae Polytrichopsida Bryopsida Poales  Liliales Coniferales Ericales MalpighialesMyrtales Malvales Fagales Fabales Rosales   Brassicales Caryophyllales  Lamiales Asterales Gentianales Ranunculales Rhizogoniales Hypnales Bryales Polytrichales Grimmiales Pottiales Cyperaceae Poaceae Liliaceae Cupressaceae Podocarpaceae Ericaceae  Salicaceae Flacourtiaceae Onagraceae Malvaceae  Nothofagaceae Fabaceae Rhamnaceae Rosaceae Brassicaceae  Caryophyllacae Polygonaceae Antirrhinaceae Asteraceae Campanulaceae Rubiaceae Papaveraceae Rhizogoniaceae Hylocomiaceae  Polytrichaceae  Grimmiaceae Pottiaceae Moraceae
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Source of rbcL DNA

• Chloroplast sequences are essentially absent from
  angiosperm pollen (Blanchard Schmidt 1995)
• The majority of the plant sequences must
  originate from locally deposited seeds, or
  somatic tissue such as the observed fine rootlets

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mtDNA 16S (88-95 bp)
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Control mtDNA region (124-129bp)
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mtDNA cyt b sequences (A, 98 bp and B, 229 bp)
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Control mtDNA region (202-203 bp)
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Control mtDNA region 234 bp
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Source of Animal mtDNA

• Unknown
• Dung is a possibility?

From Poinar et al. (2001)
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Plant Sequence Diversity(gt96 similarity)
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Frequency Herbs, Shrubs, Mosses
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Conclusions

• Diverse ancient DNA directly from soil
• (even in the absence of obvious microfossils)
• Change in plant diversity
• (following climate change)
• Change in herb/shrub dominance
• Change in Poaceae and Cyperaceae frequency
• (Pleistocene/Holocene boundary)
• Megafauna present during LGM
• DNA better preserved in permafrost than cave
  sediments
• Clutha River vegetation cover similar to
  pre-human occupation of NZ even at 600 kyr

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Perspectives

• Combined with pollen records and fossil bones
  revealing Paleobiological change
• Genetic information from archaeological records
  even in the absence of macrofossil evidence?

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DNA damage analysis

• DNA in fossil remains is known to be degraded
• Unknown to a large extent what types of damages
  accumulate
• And especially what types of damages prevents
  amplification of DNA

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DNA breaks
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Interstrand Crosslinks(Denaturation experiment)
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Rate constants
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Conclusion

• DNA in permanently frozen sediments are degraded
  by alkylation and hydrolysis, producing single
  and double stranded breaks as well as interstrand
  crosslinks
• ICL accumulate more rapidly than SSB
• SSB is generated by depurination
• The observed damage pattern indicate that DNA
  degradation result from spontaneous rather than
  exogenous processes.

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Perspectives

• Repair of ancient DNA
• Possible dating of sampels
• Determination of spontaneous accumulation of DNA
  damages in cells

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The work has been done by

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• Department of Evolutionary Biology, Zoological
  Institute, University of Copenhagen, Denmark
• Henry Wellcome Ancient Biomolecules Centre,
  Department of Zoology, University of Oxford, UK
• Department of Statistics, University of Oxford,
  UK
• Soil Cryology Laboratory, Institute for
  PhysicoChemical and Biological Problems in Soil
  Science, Russian Academy of Sciences, Russsia
• Department of Cariogenese, MDAnderson Cancer
  institute, UT

• Alan Cooper
• Anders J. Hansen
• Beth Shapiro
• Carsten Wiuf
• David A. Gilichinsky
• David Mitchell
• Eske Willerslev
• Jonas Binladen
• Lakshmi Paniker
• M. Thomas P. Gilbert
• Mike Bunce
• Regin Rønn
• Tina B. Brand

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Beringia
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Beringia Megafauna of the Late Pleistocene
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Arctic Dessert or Steppe?Why Megafauna got
Extinct?
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Traditional Approach

• Pollen analyses
• Problems
• Variation in influx rates, long distance
  dispersal, no account for vegetative growth,
  problems of taxonomic identification
• Vertebrate fossils
• Problems
• Different preservation, dating beyond carbon age
  

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Thoughts

• Is it possible to address the paleo- environment
  of Beringia by obtaining DNA directly from the
  permafrost sediments even in the absence of
  macrofossils?
• Cold conditions is critical for the long-term
  preservation of DNA (Smith et al. 2002). If plant
  or animal DNA accumulates in sediments permafrost
  must provide ideal preservation conditions