Title: Department of Evolutionary Biology
1Ancient DNA in Sediments
- Department of Evolutionary Biology
- Zoological Institute
- University of Copenhagen
2Ancient DNA Studies
3DNA from Sediments
4Sample 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
5Microscopy
- 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
6PCR 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
7Precaution, 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
-
8Important!
- 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
9Amplification 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
10Plant 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
11Source 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
12mtDNA 16S (88-95 bp)
13Control mtDNA region (124-129bp)
14mtDNA cyt b sequences (A, 98 bp and B, 229 bp)
15Control mtDNA region (202-203 bp)
16Control mtDNA region 234 bp
17Source of Animal mtDNA
- Unknown
- Dung is a possibility?
From Poinar et al. (2001)
18Plant Sequence Diversity(gt96 similarity)
19Frequency Herbs, Shrubs, Mosses
20Conclusions
- 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
21Perspectives
- Combined with pollen records and fossil bones
revealing Paleobiological change - Genetic information from archaeological records
even in the absence of macrofossil evidence?
22DNA 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
23DNA breaks
24Interstrand Crosslinks(Denaturation experiment)
25Rate constants
26Conclusion
- 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.
27Perspectives
- Repair of ancient DNA
- Possible dating of sampels
- Determination of spontaneous accumulation of DNA
damages in cells
28The 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
29Beringia
30Beringia Megafauna of the Late Pleistocene
31Arctic Dessert or Steppe?Why Megafauna got
Extinct?
32Traditional 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
33Thoughts
- 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