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Department of Evolutionary Biology

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Title: Department of Evolutionary Biology


1
Ancient DNA in Sediments
  • Department of Evolutionary Biology
  • Zoological Institute
  • University of Copenhagen

2
Ancient DNA Studies
3
DNA from Sediments
4
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
5
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

6
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

7
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

8
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

9
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

10
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
11
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

12
mtDNA 16S (88-95 bp)
13
Control mtDNA region (124-129bp)
14
mtDNA cyt b sequences (A, 98 bp and B, 229 bp)
15
Control mtDNA region (202-203 bp)
16
Control mtDNA region 234 bp
17
Source of Animal mtDNA
  • Unknown
  • Dung is a possibility?

From Poinar et al. (2001)
18
Plant Sequence Diversity(gt96 similarity)
19
Frequency Herbs, Shrubs, Mosses
20
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

21
Perspectives
  • Combined with pollen records and fossil bones
    revealing Paleobiological change
  • Genetic information from archaeological records
    even in the absence of macrofossil evidence?

22
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

23
DNA breaks
24
Interstrand Crosslinks(Denaturation experiment)
25
Rate constants
26
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.

27
Perspectives
  • Repair of ancient DNA
  • Possible dating of sampels
  • Determination of spontaneous accumulation of DNA
    damages in cells

28
The work has been done by
  •  
  • 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

29
Beringia
30
Beringia Megafauna of the Late Pleistocene
31
Arctic Dessert or Steppe?Why Megafauna got
Extinct?
32
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

33
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
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