Transcript Slide 1

Ancient DNA in Sediments
Department of Evolutionary Biology
Zoological Institute
University of Copenhagen
Ancient DNA Studies
DNA from Sediments
Sample Information
Samples
Site
Age range (B.P.)
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
Clutha River
624±50 yr
Permafrost
New Zealand
Cave sediment
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
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
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 Subclass =9
Liliopsida
Coniferopsida
Asteridae
Rosidae
Caryophyllidae
Eudicotyledon
Bryidae
Polytrichopsida
Bryopsida
Order =22
Poales
Liliales
Coniferales
Ericales
Malpighiales
Myrtales
Malvales
Fagales
Fabales
Rosales
Brassicales
Caryophyllales
Lamiales
Asterales
Gentianales
Ranunculales
Rhizogoniales
Hypnales
Bryales
Polytrichales
Grimmiales
Pottiales
Family =28
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 ursinus
S1
AJ304826
Rattus norvegicus
AJ428514
63
Volemys kikuchii
AF348082
Dicrostonyx groenlandicus
68
AY261992
clone (19 kyr) - permafrost sediment
72
Lemmus lemmus
AY261993
95
clone (19 kyr) - permafrost sediment
clone (19 kyr) - permafrost sediment
clone (19 kyr) - permafrost sediment
Oryctolagus cuniculus
AJ001588
Lepus europaeus
75
100
AJ421471
2 clones (10.4 kyr) - permafrost sediment
Capricornis crispus
U87029
clone (19 kyr) - permafrost sediment
100
96
Ovibos moschatus
U87027
Homo sapiens
Loxodonta africana
99
AF382013
AF039436
Loxodonta africana
AJ224821
clone (19 kyr) - permafrost sediment
54
97
clone (10.4 kyr) - permafrost sediment
87
Mammuthus primigenius
AF154865
Mammuthus primigenius
Z54098
9 clones (10.4, 19, and 20-30 kyr) - permafrost sediment
clone (10.4 kyr) - permafrost sediment
Dugong dugong
AY075116
Rhinoceros unicornis
X97336
92
Ceratotherium simum
Y07726
Equus hemionus (Pleistocene)
S65410
93
Equus hemionus
77
Z18645
Equus asinus
X97337
58
clone (19 kyr) - permafrost sediment
clone (19 kyr) - permafrost sediment
70
clone (19 kyr) - permafrost sediment
clone (19 kyr) - permafrost sediment
Equus caballus
X79547
8 clones (19 kyr) - permafrost sediment
Equus sp. (Pleistocene)
0.1
X86215
Control mtDNA region (124-129bp)
Vombatus ursinus
S2
AJ304826
Homo sapiens
AF347015
Ozotoceros bezoarticus
AF012572
clone (10.4 kyr) - permafrost sediment
98
100
67
Rangifer tarandus groenlandicus
AF096441
Capricornis crispus
AB055699
clone (19 kyr) - permafrost sediment
74
Ovibos moschatus
94
AY261987
Bos taurus
AB065127
Bison spp. (Pleistocene) CRS-DY-42
AY261988
83
clone (19 kyr) - permafrost sediment
78
70
Bison spp. (Pleistocene) CRS-SY-2
AF538947
clone (20-30 kyr) - permafrost sediment
96
clone (10.4 kyr) - permafrost sediment
clone (10.4 kyr) - permafrost sediment
0.1
mtDNA cyt b sequences
(A, 98 bp and B, 229 bp)
Dugong dugong
S3A
AY075116
Dugong dugong
S3B
AY075116
Loxodonta cyclotis
Elephas maximus
AF132526
100
AF132527
78
Loxodonta cyclotis
AF132529
Elephas maximus
Elephas maximus
D50844
AF132526
Loxodonta cyclotis
90
Elephas maximus
AF132527
Y13886
79
Loxodonta cyclotis
clone (8-12 kyr) - permafrost sediment
59
AF132528
clone (20-30 kyr) - permafrost sediment
Mammuthus primigenius
64
D50842
94
63
clone (8-12 kyr) - permafrost sediment
Mammuthus primigenius
AF154864
clone (8-12 kyr) - permafrost sediment
clone (20-30 kyr) - permafrost sediment
3 clones (8-12 kyr) - permafrost sediment
clone (10.4 kyr) - permafrost sediment
65
Mammuthus primigenius
D83047
0.1
clone (8-12 kyr) - permafrost sediment
Mammuthus primigenius
D50842
0.1
Control mtDNA region (202-203 bp)
clone (600 yr) Clutha River - cave sediment
S4A
68
99
clone (600 yr) Clutha River - cave sediment
Pachyornis elephantopus
AY261990
Euryapteryx curtus
AY261989
clone (1-3 kyr) Tokerau Beach - sediment inside bone
60
88
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
50
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
Megalapteryx didinus
AY261991
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
93
92
100
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
0.1
Control mtDNA region 234 bp
S4B
100
Nymphicus hollandicus
Cacatua roseicapilla
56
Nestor notabilis
100
Nestor meridionalis
Strigops habroptilus
Psephotus haematonotus
Barnardius barnardi
63
81
68
66
94
Barnardius zonarius
Psephotus varius
Northiella haematogaster
Cyanoramphus novaezelandiae
100
clone (600 yr Clutha River - cave sediment
0 .1
Source of Animal
mtDNA
Unknown
Dung 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 prehuman 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
A
H2N
N
O
-
P
N
O
Deamination
of Cytosine
N
HO
N
O
NH2
O
N
O
O
-
P
O
O
O
N
Clevage of the
phosphor backbone
O
O
N
Clevage by depurination
and ß-elimination
NH
O
O
-
P
N
O
NH2
N
O
O
O
CH3
NH
O
O
-
P
O
O
O
OH
N
O
Interstrand Crosslinks
(Denaturation experiment)
B
1.0
0.8
fICL = 0.43 + 0.49 (1 - e-0.0055 t)
DNA
fICL
Protein
0.6
R2 = 0.993
fICL = 0.43 + 0.57(1-e-0.0034 · t)
k = 0.0034 kyr-1 = 1.1 x 10-13 s-1
0.4
r2 = 0.9684
0.2
0.0
DNA
0
100
200
300
Age (kyr)
400
500
600
Rate constants
Lesion type
Time
flesion
flesion
(kyr)
DSB
10.4  300-400
ICL
10.4
T½
(sec-1)
(yr)
< 3.7 x 10-17
> 8 x 108
3.9 x 10-15
5.5x106
1.1 x 10-13
2.0 x 105
0.00013†
0.00037†
10.4  400-600
SSB
k*
0.00053‡
19
0.0016‡
10.4
0.44
19
0.49
300-400
0.85
400-600
0.87
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 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
Thoughts…
 Is it possible to address the paleoenvironment of Beringia by obtaining
DNA directly from the permafrost
sediments even in the absence of
macrofossils?
 Cold conditions is critical for the longterm preservation of DNA (Smith et al.
2002). If plant or animal DNA
accumulates in sediments permafrost
must provide ideal preservation
conditions