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Department of Evolutionary BiologyZoological InstituteUniversity of Copenhagen
Ancient DNA in Sediments
Ancient DNA Studies
DNA from Sediments
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 10.425±45 yr
2/01/4.8 Laptev Sea coast, Cape Bykovskii 18.980±70 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 624±50 yr
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
Microscopy
PCR Based Analyses
4 x 0.25gww soil FAST PREPDNA extraction/purificationPCR (“universal”/”specific”
primers for rbcL/mtDNA)CloningSequencing BLAST (GenBank)/phylogenetic
analysis
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
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
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
Plant identifications (multiple GenBank sequences showing >96%
similarity to the clones; reproducibility confirmed by a bootstrap test )
Class or Sub-class =9
Order =22 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
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
mtDNA 16S (88-95 bp)Vombatus ursinusAJ304826
Dugong dugongAY075116
Homo sapiensAF382013
Oryctolagus cuniculusAJ001588
Lepus europaeusAJ421471
2 clones (10.4 kyr) - permafrost sediment100
100
Volemys kikuchiiAF348082
Dicrostonyx groenlandicusAY261992
54
93
92
75
63
68
72
95
Loxodonta africanaAF039436Loxodonta africanaAJ224821
99
96
97
Rhinoceros unicornisX97336Ceratotherium simumY07726
87
58
77
70
clone (19 kyr) - permafrost sediment
Lemmus lemmusAY261993
clone (19 kyr) - permafrost sediment
clone (19 kyr) - permafrost sediment
clone (19 kyr) - permafrost sediment
Mammuthus primigeniusAF154865
Mammuthus primigeniusZ54098
clone (10.4 kyr) - permafrost sediment
clone (10.4 kyr) - permafrost sediment
clone (19 kyr) - permafrost sediment
clone (19 kyr) - permafrost sediment
9 clones (10.4, 19, and 20-30 kyr) - permafrost sediment
Equus hemionus (Pleistocene)S65410
Equus hemionusZ18645
Equus asinusX97337
Equus caballusX79547
Equus sp. (Pleistocene)X86215
clone (19 kyr) - permafrost sediment
clone (19 kyr) - permafrost sediment
clone (19 kyr) - permafrost sediment
clone (19 kyr) - permafrost sediment
8 clones (19 kyr) - permafrost sediment
0.1
Rattus norvegicusAJ428514
Capricornis crispusU87029
Ovibos m
Control mtDNA region (124-129bp)
Vombatus ursinusAJ304826
Homo sapiensAF347015
Ozotoceros bezoarticusAF012572
Rangifer tarandus groenlandicusAF096441
clone (10.4 kyr) - permafrost sediment
100
98
clone (19 kyr) - permafrost sediment
Capricornis crispusAB055699
Ovibos moschatusAY261987
74
67
Bos taurusAB065127
Bison spp. (Pleistocene) CRS-DY-42AY261988
clone (19 kyr) - permafrost sediment
Bison spp. (Pleistocene) CRS-SY-2AF538947
70
96
78
83
94
clone (20-30 kyr) - permafrost sediment
clone (10.4 kyr) - permafrost sediment
clone (10.4 kyr) - permafrost sediment
0.
mtDNA cyt b sequences (A, 98 bp and B, 229 bp)
Dugong dugongAY075116
Loxodonta cyclotisAF132527
Loxodonta cyclotisAF132528
79
Elephas maximusAF132526
Elephas maximusD50844
100
Mammuthus primigeniusD50842
clone (20-30 kyr) - permafrost sediment
clone (20-30 kyr) - permafrost sediment
clone (10.4 kyr) - permafrost sediment
Mammuthus primigeniusD83047
65
64
0
Dugong dugongAY075116
Loxodonta cyclotisAF132527
Loxodonta cyclotisAF132529
78
Elephas maximusAF132526
Elephas maximusY13886
90
clone (8-12 kyr) - permafrost sediment
Mammuthus primigeniusAF154864
Mammuthus primigeniusD50842
clone (8-12 kyr) - permafrost sediment
clone (8-12 kyr) - permafrost sediment
clone (8-12 kyr) - permafrost sediment
63
94
59
3 clones (8-12 kyr) - permafrost sediment
0.
Control mtDNA region (202-203 bp)
clone (1-3 kyr) Tokerau Beach - sediment inside bone
clone (1-3 kyr) Tokerau Beach - sediment inside bone
clone (1-3 kyr) Tokerau Beach - sediment inside bone
clone (1-3 kyr) Tokerau Beach - sediment inside bone
clone (1-3 kyr) Tokerau Beach - sediment inside bone
clone (1-3 kyr) Tokerau Beach - sediment inside bone
clone (1-3 kyr) Tokerau Beach - sediment inside bone
clone (1-3 kyr) Tokerau Beach - sediment inside bone
clone (1-3 kyr) Tokerau Beach - sediment inside bone
clone (1-3 kyr) Tokerau Beach - sediment inside bone
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
clone (600 yr) Clutha River - cave sediment
Euryapteryx curtusAY261989
Pachyornis elephantopusAY261990
Megalapteryx didinusAY261991
60
50
99
68
100
93
92
88
Control mtDNA region 234 bp
Psephotus varius
0.1
Nymphicus hollandicus
Cacatua roseicapilla
Nestor notabilis
Nestor meridionalis
Strigops habroptilus
Psephotus haematonotus
Barnardius barnardi
Barnardius zonarius
Northiella haematogaster
Cyanoramphus novaezelandiae
clone (600 yr Clutha River - cave sediment
100
100
56
68
63
81
66
100
94
Source of Animal mtDNA
UnknownDung is a possibility?
From Poinar et al. (2001)
Plant Sequence Diversity
(>96% similarity)
Frequency; Herbs, Shrubs, Mosses
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
Perspectives
Combined with pollen records and fossil bones revealing Paleobiological change
Genetic information from archaeological records even in the absence of macrofossil evidence?
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
DNA breaks
N
NH
O
O
CH3
N
NH
N
N
NH2
O
N
N
NH2
O
N
N
N
N
NH2
OO
O
OH
O-
O
P
OO
O
O-
O
P
OO
O
O-
O
P
OO
OH
O-
O
P
Clevage by depurinationand ß-elimination
Clevage of thephosphor backbone
Deaminationof Cytosine
Interstrand Crosslinks(Denaturation experiment)
DNA
DNAProtein
B
fICL = 0.43 + 0.49 (1 - e-0.0055 t)
R2 = 0.993
Age (kyr)
0 100 200 300 400 500 600
f ICL
0.0
0.2
0.4
0.6
0.8
1.0
fICL = 0.43 + 0.57(1-e-0.0034 · t)
k = 0.0034 kyr-1 = 1.1 x 10-13 s-1
r2 = 0.9684
Rate constants
Lesion type
Time
(kyr)
flesion
flesion
k*
(sec-1)
T½
(yr)
DSB 10.4 300-400 0.00013†
10.4 400-600
0.00037† < 3.7 x 10-17 > 8 x 108
SSB 10.4 0.00053‡
19 0.0016‡ 3.9 x 10-15 5.5x106
ICL 10.4 0.44
19 0.49
300-400 0.85
400-600 0.87
1.1 x 10-13 2.0 x 105
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.
Perspectives
Repair of ancient DNA
Possible dating of sampels
Determination of spontaneous accumulation of DNA damages in cells
The work has been done by:
• 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
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
Beringia
Beringia Megafauna of the
Late Pleistocene
Arctic Dessert or Steppe?Why Megafauna got
Extinct?
Traditional Approach
Pollen analysesProblems: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
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